Fecundation  in  Plants 


BY 


DAVID  M.  MOTHER,  Ph.  D., 


Professor  of  Botany  in  Indiana  University 


Published  by  the  Carnegie  Institution 

of  Washington 

1904 


msM^mmjB^Sl^j^^m^ 


http7/wWw.archive.org/detalis/fecuhciaMiiif^ 


Fecundation  in  Plants 


BY 


DAVID  M.  MOTHER,  Ph.  D.. 


Professor  of  Botany  in  Indiana  University 


Published  by  the  Carnegie  Institution 

of  Washington 

1904 


Carnegie  Institution  of  Washington, 
Publication  No.  15. 


Press  of  Gibson  Bros., 
Washington,  D.  C. 


PREFACE. 


This  volume  presents  the  subject  of  fecundation  in  the  vegetable 
kingdom  by  the  discussion  of  concrete  cases,  selecting  from  the  great 
groups  of  plants  certain  typical  representatives  in  which  the  sexual 
process  seems  to  have  been  most  thoroughly  investigated.  In  the 
introductory  chapter  I  have  discussed  typical  processes  of  nuclear 
division  and  cell-formation,  especially  in  spore  mother-cells,  together 
with  a  few  topics  dealing  with  certain  phenomena  of  the  cell  and  the 
significance  of  sexuality.  This  is  considered  necessary  to  a  better 
understanding  of  sexual  reproduction,  for  problems  of  sexuality,  like 
problems  of  evolution,  have  in  late  years  become  reduced  to  problems 
of  the  cell,  and,  since  the  nucleus  plays  by  far  the  most  important 
part  in  fecundation,  I  am  tempted  to  say  to  problems  of  the  nucleus. 

The  processes  leading  to  the  development  and  differentiation  of  the 
gametes  have  been  regarded  as  of  prime  importance,  and  they  have 
therefore  received  emphasis.  Whenever  the  subsequent  history  of  the 
fecundated  egg  has  been  followed  to  any  extent  this  has  been  done,  as 
in  the  Ascomycetes  and  Floride^^  to  show  the  relation  between  the 
real  sexual  process  and  the  vegetative  fusion  of  nuclei  which  has  been 
confused  with  the  sexual  act,  and,  as  in  the  Desmids,  for  the  sake  of 
pointing  out  certain  nuclear  phenomena  that  take  place  during  the 
germination  of  the  zygote  with  similar  phenomena  just  preceding  the 
sexual  act  in  the  Diatoms.  Processes  which  are  purely  morphological 
are  assumed  or  dealt  with  very  briefly. 

In  grouping  the  representative  types  into  the  several  chapters  I  have 
had  in  mind  no  particular  theory  of  the  evolution  of  sexuality,  but 
merely  the  idea  of  the  evolution  of  the  plant  kingdom  and  the  corre- 
sponding differentiation  of  the  sexual  organs  and  cells  accompanying 
this  evolution  in  the  groups  of  plants  themselves. 

The  chapters  dealing  with  the  lower  plants  in  which  the  develop- 
ment of  the  gametes  is  not  known  from  a  modern  cytological  standpoint, 
and  in  which  the  behavior  of  the  sexual  nuclei  in  the  fusion  of  the 
gametes  has  not  been  followed — have  been  made  as  brief  as  possible. 
For  a  similar  reason  the  mosses  and  liverworts  have  been  omitted 
entirely. 


iv  PREFACE. 

No  attempt  has  been  made  to  discuss  the  numerous  theories  bearing 
upon  the  subject.  Whenever  theoretical  matters  are  touched  upon  the 
object  has  been  chiefly  to  suggest  probable  lines  of  investigation.  I 
have  not  hesitated,  however,  to  express  my  own  opinion  in  all  cases 
in  which  my  special  field  of  study  has  given  me  a  first-hand  knowledge 
of  the  subject-matter. 

To  designate  the  sexual  process  which  consists  in  the  fusion  of  sex- 
ually differentiated  cells,  or  gametes,  and  especially  the  fusion  of  their 
nuclei,  the  term  fecundation  has  been  used  instead  oi  fertilization — 
fecundation  being  the  equivalent  of  the  German  Befruchtung  and 
the  Yrenchfecondation. 

It  has  been  necessary,  of  course,  to  copy  numerous  figures  from  the 
papers  of  other  investigators,  but  in  every  case  due  credit  is  given. 

In  the  citation  of  literature  in  the  text  the  author  is  referred  to  by 
the  year  in  which  his  work  was  published.  No  attempt  has  been  made 
to  g^ve  a  complete  bibliography,  and  no  doubt  many  valuable  refer- 
ences have  been  omitted. 

The  author  is  indebted  to  Professors  W.  Belajeff,  H.  O.  Juel, 
F.  Oltmanns,  S.  Ikeno,  and  to  Dr.  H.  Klebahn,  Dr.  A.  H.  Trow, 
Dr.  H.  Wager,  Dr.  S.  Hirase,  and  Dr.  V.  H.  Blackman,  for  re- 
prints of  their  papers,  from  many  of  which  illustrations  have  been 
borrowed,  and  especially  to  Professor  R.  A.  Harper  for  helpful 
suggestions. 

David  M.  Mottier. 

Indiana  University,  August^  igo2. 


CONTENTS. 


Chapter  I. — Introduction. 


Pagk. 

Nuclear  division,    .... 

. 

2-30 

Karyokinesis  in  cells  of  the  lower  plants  in 

which  centrospheres  are 

developed, 

. 

2-10 

Dictyota,        .... 

. 

2 

Erysiphe, 

. 

7 

Mitosis  in  pollen  mother-cells, 

. 

11-30 

The  first  or  heterotypic  mitosis, 

. 

11-26 

Resting  nucleus  and  the  development  of  the  chromatin  spirem 

II 

Development  of  the  spindle. 

15 

Chromosomes,    . 

. 

17 

Metakinesis, 

20 

The  anaphase,    . 

. 

22 

The  telophase, 

23 

The  nucleolus,     . 

. 

25 

The  second,  or  homotypic  division, 

27-31 

Cell  division, 

. 

31-44 

The  type  of  the  higher  plants. 

31 

Free  cell-formation. 

33 

Cell-cleavage,      .             .             .             • 

36 

Cell-division  in  Dictyota  and  Stypocaulon, 

. 

41 

The  centrosome  and  the  blepharoplast. 

44 

The  significance  of  the  sexual  process  and  the 

numerical  reduction  of 

the  chromosomes, 

. 

49-60 

Chapter  II. — Fecundation;  Motile  Isogametes. 

Ulothrix  and  Hydrodictyon,   ......  61-65 

Copulation  of  gametes,     .......        65 

Ectocarpus,  .......  "5 


Chapter  III.— Fecundation;  Non-Motile  Isogametes. 

Spirogyra,  ....•••• 

Sporodinia,     ...••••• 

Closterium  and  Cosmarium,  ...... 

Diatoms  (Rhopalodia,  Cocconeis),     ..... 

Basidiobolus,         .....••• 


67 
71 
71 
73 
76 


VI  CONTENTS. 

Chapter  IV. — Fecundation  ;  Heterogametes. 

Sphseroplea,  ........  79 

Fucaceae  (Fucus,  Halidrys),  ......         84 

Volvox,           ........  88 

OEdogonium,         ........         89 

Coleochaete,                .            .            .            .            .            .            .  91 

Vaucheria,  ........         94 

Albugo  (Cystopus),     .......  96 

Achlya  and  Saprolegnia,               .             .             .             .             .  .102 

Chapter  V. — Type  of  the  Ascomycetes  and  Rhodophyce.(E  . 

Sphaerotheca,              .             •             •             •             .             .             .  108 

Pyronema,             .             •             •             •             •             .             .  iir 

Batrachospermum,     .......  116-119 

Dudresnya,           .......  1 19-126 

CoUema,        ........  126-128 

Chapter  VI. — Archegoniates. 

.Pteridophyta,        •            •          •  •             ....             .             .  i2g 

The  spermatozoid,           ......  130-136 

The  egg-cell  and  fecundation,            ....  136-142 

Gymnosperms,           .......  j^2 

Cycas,  Zamia,  and  Ginkgo,                .             .             .             .  j^2 

The  male  gametophyte  and  the  development  of  the  sperma- 

tozoids,     .......  142-155 

The  archegonium,           .....  156-158 

Fecundation,            ......  158-163 

Pinus,            .......  j5. 

The  male  and  female  gametophytes,            .             .             .  163-164 

Fecundation,      ......  165-168 

Chapter  VII. — Angiosperms. 

The  embryo-sac,  or  female  gametophyte,      ....  169-174 

The  male  gametophyte,                .....  174-176 

The  fusion  of  male  and  egg-nucleus,                 ....  176-177 

The  fate  of  the  second  male  nucleus  in  the  embryo-sac,               .  177-180 

Bibliography, 1 81-187 


INDEX. 

Page. 

Abies              ...... 

156 

Achlya     ....... 

102-107 

Adiantum      ...... 

136 

Albugo    ....... 

96-100 

Aspidium       ...... 

136 

Basidiobolus        ...... 

76-78 

Batrachospermum     .       "■     • 

116-119 

Callithamnion     ...... 

I 19-124 

Cell-cleavage  in  Synchitrium  discipens 

36-38 

Pilobolus  crystallinus 

38-41 

Cell-division  in  higher  plants 

31-33 

Dictyota  and  Stypocaulon 

4'-43 

Cell- formation,  free,  in  Erysiphe  communis 

33-35 

Lachnea  scutellata 

. 

35 

Centrosome,  in  Dictyota      .... 

3-7 

Erysiphe             .             .             .             . 

8-10 

Centrosome  and  Blepharoplast 

44-49 

Cephalotaxis        ..... 

157' 

Chara             ...... 

135-136 

Chromosomes  in  tetraspore  mother-cell  of  Dictyota     . 

5-6 

ascus  of  Erysiphe 

8-1 1 

pollen  mother-cells  of  Lilium 

17-31 

Podophyllum 

17-31 

Tradescantia 

17-31 

Significance  of  numerical  reduction 

49-60 

Closterium           ..... 

71 

Cocconeis      ...... 

75 

Coleochsete          ... 

91-93 

CoUema         ...... 

126-128 

Cosmarium          ...... 

71,  72 

Cycas             ...... 

142-149,  156,  1 

57,  163,  166 

Cystopus  Csee  Albugo). 

Dasya       ...... 

124 

Diatoms         ...... 

73-76 

Dictyota                ..... 

2-6,  26 

Dudresnya     ...... 

119-125 

Ectocarpus            ..... 

65,  66 

Equisetum     ...... 

135 

Erysiphe               ..... 

7-10 

Fucus              ...... 

84-88 

Ginkgo    ...... 

149-155. 

162,  163,  166 

Gloecosiphonia          ..... 

124 

Gnetum                 ..... 

.      168,  173 

Gymnogramme         ..... 

130-132 

Halidrys                ..... 

8s 

Helleborus                 ..... 

.    12,  158, 

169-171,  173 

Hydrodictyon      .             .             •            .            . 

63-65 

INDEX. 


Karyokihesis  (see  Mitosis). 

Laboulbeniaceae 

Larix        .... 

Lilium  : 

Mitosis  in  pollen  mother-cells 

Development  of  mitotic  spindle  in  pollen  mother-cells 

Behavior  of  chromosomes  in  pollen  mother-cells 

Nucleolus      ..... 

Second  or  homotypic  mitosis  in  pollen  mother-cells 

Embryo-sac  and  Fecundation 

Fate  of  second  male  nucleus  in  embryo-sac 

Marsilia  ...... 

Mitosis  in  Dictyota 
Erysiphe 
pollen  mother-cells 

Monotropa 

Nemalion 

Nucleolus,  discussion  of 

CEdogonium 

Onoclea  . 

Peperomia     . 

Peronospora 

Picea 

Pilularia 

Pinus 
'Physcia    ...... 

Podophyllum : 

Resting  nucleus  of  pollen  mother-cell 

Nature  of  nuclear  membrane 

Behavior  of  chromosomes  in  pollen  mother-celt 

Pteridophyta        ..... 

Pyronema 

Pythium 

Rhopalodia  gibba 

Saprolegnia 

Sphaeroplea  . 

Sphaerotheca 

Spirogyra 

Sporodinia 

Synapsis 

Tradescantia  virginica  : 

Behavior  of  chromosomes  in  pollen  mother-cell 
Second  or  homotypic  mitosis  in  pollen  mother-cell 

Tsuga 

Tulipa 

Ulothrix 

Vaucheria 

Vicia  faba 

Volvox 

Zamia 

Zea  mays 


133. 


130- 


H9-ISS,  1 


Page. 

126 
158,  170-171 

11-30 

15-16 

17-24 

25 

27-30 

169-177 

177-178 

134^  135 

2-7 

7-1 1 

11-29 

177 

119,    131 

25,  26 

89-91 

'33.  136,  138-141 
173 

lOI 

163 

142 

156,  163-168 

128 

II,  12 

13.  24 

18,  22 

129-142 

111-116 

lOI 

73>  75,  76 
102,  107 

79-84 

108-111 

26,  67-70,  168 

71 

13 

.     18,  19,  22 
27,  29 
163,  165,  166,  167 
178 
61,  62,  65 
94.95 
25 
88 
57-161,  163,  166 
25,  178 


FECUNDATION  IN  PLANTS. 


CHAPTER  I.— INTRODUCTION. 

The  processes  of  nuclear  division  and  cell-formation  are  so  closely 
associated  with  sexual  cells  and  their  development  that  an  adequate 
understanding  of  these  cells  is  impossible  without  a  definite  and 
thorough  knowledge  of  the  processes  involved  in  their  development. 
Our  interpretations  of  the  significance  of  the  sexual  process  and  the 
phenomena  of  heredity  in  all  organisms  will  be  more  lasting  and  help- 
ful as  scientific  knowledge  if  these  interpretations  or  doctrines  are 
based  upon  a  well-connected  phylogenetic  series  of  the  most  funda- 
mental facts.  Perhaps  no  other  field  of  research  has  been  more 
helpful  during  the  past  quarter  of  a  century  in  enabling  the  biologist 
to  gain  a  deeper  and  more  far-reaching  knowledge  of  the  physical 
basis  of  heredity  than  the  study  of  mitosis,  especially  in  reproductive 
cells.  The  division  of  the  nucleus  naturally  suggests  the  division  of 
the  cell,  or  the  process  by  which  new  cells  are  formed  from  a  mother- 
cell,  and  the  study  of  cell-formation  in  very  recent  years,  especially 
among  the  lower  plants,  has  not  only  wrought  almost  a  revolution 
in  our  knowledge  of  the  processes  here  involved,  but  has  also  furnished 
new  criteria  for  determining  relationships  and  probable  lines  of  descent. 

It  is  deemed  necessary,  therefore,  to  introduce  the  subject  of  sexual 
reproduction  in  plants  by  a  brief  presentation  of  the  typical  processes 
of  nuclear  ahd  cell-division  in  both  the  lower  and  higher  forms.  In 
doing  so  these  processes  will  be  described  in  a  few  of  those  forms 
which  have  been  subjected  to  a  critical  study  by  means  of  the  most 
improved  methods  and  instruments.  The  processes  described  will  be 
confined  largely,  though  not  exclusively,  to  spore  mother-cells. 

The  division  of  the  nucleus  and  of  the  cell  presents  generally  three 
processes,  the  development  of  the  karyokinetic  spindle,  the  behavior 
of  the  chromatin,  and  the  formation  of  the  cell-plate  or  new  plasma 
membrane.  This  division  is  made  merely  for  the  sake  of  convenience, 
as  it  is  not  implied  that  three  distinct  or  separate  processes  are 
necessarily  involved,  although  the  development  of  the  plasma  mem- 
brane in  many  cases  has  apparently  little  or  no  connection  with  the 


2  ''  INTRODUCTION. 

division  of  the  nucleus.  The  first  two  of  these  processes  will  be  dis- 
cussed under  nuclear  division^  while  the  third  will  be  dealt  with  in 
connection  with  cell-formation. 

NUCLEAR  DIVISION. 

KARYOKINESIS   IN   CELLS   OF   THE   LOWER   PLANTS   IN   WHICH 
CENTROSOxMES  AND  CENTROSPHERES  ARE  DEVELOPED. 

At  present  there  are  recognized  two  types  of  development  of  the 
karyokinetic  spindle.  In  one  the  spindle  arises  through  the  instru- 
mentality of  individualized  dynamic  centers  or  centrospheres,  as  in 
certain  Thallophyta  and  Liverworts ;  in  the  other,  it  is  developed 
wholly  independently  and  in  the  absence  of  any  such  centers,  as,  for 
example,  in  the  higher  plants.  We  speak  of  types  of  spindle  develop- 
ment in  this  connection  also  for  the  sake  of  convenience,  since  centro- 
spheres have  not  been  found  in  connection  with  the  development  of 
the  spindle  in  all  Thallophytes ;  but  the  author  does  maintain  that 
centrospheres  have  not  been  demonstrated  to  occur  in  any  plant 
above  the  Bryophytes,  and  that  in  the  Angiosperms  such  structures 
do  not  in  all  probability  exist. 

As  illustrating  the  development  of  the  spindle  in  which  centro- 
spheres are  present,  the  tetraspore  mother-cell  in  Dictyota  dichotoma 
will  be  selected  from  the  algae  and  the  mother-cell  of  the  ascus  in 
Erysiphe  from  the  fungi. 

It  is  not  considered  necessary,  nor  conducive  to  any  better  under- 
standing of  the  facts  presented  here,  to  enter  into  any  lengthy  dis- 
cussion concerning  the  structure  of  the  firmer  framework  of  the 
cytoplasm.  The  consensus  of  opinion  now  is  that  the  firmer  substance 
of  cytoplasm  consists  of  either  a  reticulum  of  fibrillae  or  of  an  alveolar 
or  foam  structure  (Waben  of  German  literature)  and  that,  in  many 
cells,  these  two  structures  intergrade  into  one  another. 

DICTYOTA. 

The  cytoplasm  of  the  tetraspore  mother-cell  of  Dictyota  dichotoma 
during  the  preparation  for  nuclear  division  presents  two  well-defined 
portions,  the  kinoplasm,  which  is  always  associated  with  the  nucleus 
and  plays  the  most  important  r61e  in  the  karyokinetic  process,  and  the 
remaining  alveolar  portion.  Numerous  chloroplasts  are  also  present. 
The  first  indication  of  mitosis  is  the  appearance,  on  opposite  sides 
of  the  nucleus,  of  two  large  sharply  defined  asters  of  kinoplasmic 
fibers  radiating  from  a  rod-shaped  body,  which  is  often  slightly  bent, 
lying  either  close  to  the  nuclear  membrane  or  at  some  little  distance 
from  it  (Fig.  i ,  A) .     The  rod-shaped  body  is  the  centrosome^  which 


NUCLEAR    DIVISION. 


together  with  the  kinoplasmic  radiations  constitutes  the  centrosphere. 
The  planes  of  the  longitudinal  axes  of  the  centrosomes  may  be  parallel 
or  form  various  angles  with  each  other.  In  Fig.  i,  B,  the  centrosome 
at  the  upper  side  of  the  nucleus  is  seen  from  the  side,  the  lower  from 


Fig.  X. — First  mitosis  in  tetraspore  mother-cell  of  Dictyota  dichotonta. 
A,  B,  early  prophase  ;   the  well-developed  centrospheres  are  on  diametrically  opposite  sides  of  nuclei. 

C,  the  kinoplasmic  fibers  have  begun  to  enter  the  nucleus  to  form  the  spindle  and  the  chromosomes  are 

being  differentiated. 

D,  numerous  spindle  fibers  have  entered  the  nucleus,  and  the  chromosomes  are  collected  in  the  equa- 

torial  region. 

the  end.  Viewed  from  the  pole,  the  centrosome  is  always  rod-shaped. 
The  kinoplasmic  fibers  radiate  in  all  directions  into  the  cytoplasm 
where  they  pass  over  into  the  framework  of  the  same.  On  the  side 
next  the  nucleus  they  may  run  parallel  with  its  wall  for  some  dis- 


A  INTRODUCTION. 

tance.  Near  the  nucleus  the  cytoplasm  is  more  granular,  with  smallet 
meshes.  It  is  more  nearly  a  thread-like  net-work  than  alveolar  in 
structure,  and  appears  with  differential  staining  as  kinoplasm.  This 
very  fine  granular  thread-work  often  extends  in  among  the  radiations 
of  the  centrosphere. 

The  resting  nucleus  shows  a  large  vacuolated  nucleolus  and  a  fine 
linin-reticulum  with  rather  large  meshes,  upon  which  are  arranged 
small  and  nearly  uniform  granules,  all  of  which  do  not  react  as 
chromatin.  With  the  advance  of  karyokinesis,  the  chromatin  begins 
to  collect  into  larger  and  somewhat  irregular  masses  that  finally  become 
the  chromosomes.  There  is  not  developed,  as  in  vegetative  cells  of 
this  plant,  a  regular  and  uniform  chromatin  spirem  or  ribbon.  The 
nucleolus  becomes  more  vacuolated  and  soon  disappears.  The  nuclear 
cavity  presents  a  more  granular  appearance,  the  granules  staining 
more  densely. 

The  kinoplasmic  fibers  now  penetrate  the  membrane  of  the  nucleus 
and  enter  its  cavity,  while  at  the  same  time  the  polar  radiations  seem 
to  diminish  in  number  (Fig.  i,  C).     On  entering  the  cavity  some  of 
the  fibers  proceed  in  advance  of  the  others.     Some  pass  straight  to- 
ward the  center  of  the  nucleus,  while  others  diverge  toward  the  sides. 
As  these  fibers  approach  from  opposite  sides  of  the  nucleus,  they  tend 
to  collect  the  chromosomes  into  an  irregular  mass  in  the  equatorial 
region,  where  they  finally  form  the  nuclear  plate  (Fig.  i,  D).     Cer- 
tain of  these  fibers  coming  from  opposite  sides  seem  to  unite  at  their 
ends  to  form  the  continuous  spindle  fibers  which  extend  from  pole  to 
pole ;  others  fasten  themselves  to  the  chromosomes,  and  still  others 
diverge  toward  the  nuclear  membrane  in  the  equatorial  region  (Fig.  2, 
E).     In  the  mature  spindle,  therefore,  the  fibers  present  the  following 
orientation :   those  radiating  from  the  poles,  the  continuous  spindle 
fibers  extending  uninterruptedly  from  pole  to  pole,  those  running  from 
the  poles  to  the  chromosomes,  and  the  fibers  which  diverge  from  the 
poles  toward  the  equatorial  region  and  end  in  the  cytoplasm  (Fig.  2,  F) . 
The  nuclear  membrane  in  the  tetraspore  mother-cell  of  Dictyota 
disappears  very  gradually  during  the  process  of  karyokinesis,  often 
persisting  at  the  sides  when  the  spindle  is  mature  (Fig.  2,  F).     It  begins 
to  disappear  at  the  poles  as  soon  as  the  fibers  enter  the  nuclear  cavity, 
and  by  the  time  the  anaphase  is  reached  no  part  of  the  membrane  can 
be  distinctly  seen.     Thus  the  spindle,  with  the  exception  of  the  polar 
radiations,  lies  within  the  nuclear  cavity,  its  fibers,  however,  being 
largely  of  cytoplasmic  origin.     To  what  extent  any  nuclear  substance 
contributes  to  the  formation  of  the  spindle  is  ditficult  to  determine. 
On  the  disappearance  of  the  nucleolus,  numerous  granules  appear  in 


NUCLEAR    DIVISION.  5 

the  nucleus,  which  stain  deeply,  closely  resembling  the  chromatin 
granules.  In  the  meantime  the  chromosomes  increase  in  size,  and  it 
seems  reasonable  to  suppose  that  the  nucleolar  substance  contributes 
materially  to  their  growth.  The  development  of  the  nucleolus  in  the 
daughter  nucleus  and  its  behavior  during  the  following,  or  second 
mitosis,  seem   to  strengthen  this  theory.     The  chromosomes,  when 


Fig.  2. — Spindle  and  telophase  of  first  mitosis  in  the  tetraspore  mother-cell  of  Dictyota  dichotoma. 

E,  spindle  nearly  mature ;    nuclear  rafembrane  has  disappeared  at  poles. 

F,  mature  spindle ;   the  small  lumpy  chromosomes  are  regularly  arranged  in  equatorial  plate ;  nuclear 

membrane  persists  at  sides. 

G,  dau^ter  nuclei  still  connected  by  strand  of  connecting  fibers ;  at  poles  of  each  nucleus  is  a  well- 
developed  centrosphere. 

arranged  in  the  equatorial  plate,  appear,  especially  when  crowded  to- 
gether— a  phenomenon  of  frequent  occurrence — as  rounded  lumps 
(Fig.  2,  E,  F).  A  careful  study  in  favorable  cases  shows  clearly  that 
each  chromosome  is  either  in  the  shape  of  a  ring,  or  so  contracted  as 
to  leave  scarcely  any  central  space,  such,  for  example,  as  occurs  in 
some  higher  plants  [Podophyllum^  Helleborus) .     In  such  cases  each 


6  INTRODUCTION. 

segment  or  daughter  chromosome  forms  one-half  of  the  ring,  or 
each  maybe  in  the  form  of  a  short,  thick  U  (Fig.  2,  F).  Sixteen 
chromosomes,  the  reduced  number,  are  present  in  the  first  mitosis. 

While  on  the  way  to  the  poles  the  daughter  chromosomes  sometimes 
fuse  with  one  another  to  form  large  masses.^  This  is  especially  so  in 
the  second  mitosis. 

In  the  construction  of  the  daughter  nuclei,  one  or  more  larger  masses 
of  chromatin  are  formed  by  the  chromosomes ;  a  nucleolus  appears 
near  the  chromatin  mass  or  masses,  and  a  nuclear  membrane  is  laid 
down  (Fig.  2,  G).  The  membrane  is  unquestionably  formed  through 
the  agency  of  the  kinoplasmic  fibers.  The  centrosomes  increase  in 
size,  and  the  polar  radiations  are  more  distinct  than  in  the  spindle 
stage.  The  connecting  fibers  usually  persist  until  the  nuclear  mem- 
brane is  present,  but  a  little  later  they  disappear  entirely.  The  chro- 
matin mass,  gradually  becoming  less  dense,  soon  disintegrates,  and 
each  daughter  nucleus  passes  into  the  resting  condition  (Fig.  2,  G). 

From  the  preceding  it  will  be  seen  that  each  daughter  nucleus  is 
provided  with  one  centrosome,  but  in  the  first  mitosis  the  centrosomes 
could  not  be  made  out  until  they  were  on  opposite  sides  of  the  nucleus 
and  provided  with  radiations.  The  question  naturally  arises :  Does 
the  centrosome  divide  to  give  rise  to  the  two  daughter  centrosomes .'' 

Swingle  ('97)?  who  has  traced  the  persistence  of  the  centrosome 
through  several  successive  generations  of  vegetative  cells  in  Stypo- 
caulon^  one  of  the  Phceophycece,  found  that  a  division  of  the  centro- 
some takes  place,  and  Strasburger  ('97)  arrives  at  the  same  conclusion 
as  regards  Fucus.     This  is  the  generally  accepted  view. 

We  shall  trace  the  early  development  of  the  spindle  in  the  second 
mitosis  in  the  tetraspore  mother-cell  in  order  to  see  what  evidence  is 
furnished  by  Dictyota  toward  the  solution  of  this  problem. 

During  the  reconstruction  of  the  daughter  nucleus  (Fig.  3,  H) 
two  rod-shaped  centrosomes,  each  with  its  radiations,  were  observed 
close  together,  and  in  such  a  position  as  to  form  a  wide  V,  giving  the 
impression  that  a  longitudinal  division  of  the  single  centrosome  had 
taken  place.  The  manner  in  which  a  cluster  of  radiations  is  attached 
to  each  daughter  centrosome  seems  to  lend  weight  to  this  conclusion. 

The  daughter  centrosomes  now  separate,  moving  along  the  nuclear 
membrane,  but  they  do  not,  as  in  the  first  mitosis,  traverse  an  angular 
distance  of  180°  before  the  formation  of  the  spindle  begins  (Fig.  3, 
I,  K) .  The  development  of  the  spindle  is  the  same  as  in  the  first 
mitosis,  as  Fig.  3,  I,  J,  K,  L,  will  clearly  show. 

In  other  brown  algse,  so  far  as  known  (Swingle  '97,  Strasburger  '97) , 

'  This  massing  of  the  chromosomes  may  not  occur  in  all  cases. 


NUCLEAR    DIVISION. 


the  development  of  the  karyokinetic  spindle  in  both  vegetative  and 
reproductive  cells  agrees  essentially  with  that  described  for  Dictyota. 
In  the  diatoms  the  development  of  the  spindle  as  described  by 
Lauterborn  ('96)  is  singular  and  without  parallel  in  the  plant  king- 
dom. According  to  this  author,  the  spindle  develops  directly  from 
the  centrosome  by  a  division  of  the  same  or  by  budding.  We  shall 
refer  to  this  phenomenon  beyond  in  the  section  dealing  especially  with 
the  centrosome.  In  the  red  algae  the  development  of  the  karyokinetic 
figure  is  known  somewhat  in  detail  only  in  Corallina  officinalis.  In 
this  plant,  Davis  ('98)  finds  that  the  spindle  arises  through  the  agency 


Fig.  3. — Second  mitosis  in  tetraspore  mother-cell  q{ Dictyota. 
H-K,  prophase,  showing  origin  of  spindle.  L,  a  nearly  mature  spindle. 

of  centrospheres  which  undergo  a  great  change  in  size  during  mitosis. 
The  persistence  of  these  bodies  was  not  followed  from  one  cell  genera- 
tion to  the  next.  The  paucity  of  our  knowledge  of  nuclear  division 
in  the  red  algae  precludes  any  further  mention  of  the  subject  in  this 
group  of  plants.  So  far  as  is  known  to  the  author,  no  centrospheres 
or  centrosomes  have  been  authentically  observed  in  the  green  algae. 

ERVSIPHE  COMMUNIS, 

For  the  fungi,  the  most  accurate  and  complete  account  of  karyoki- 
nesis  is  to  be  found  in  the  classical  work  of  Harper  ('97)  on  certain 
Ascomycetes.  As  an  illustration  of  the  process  in  this  group  of  fungi, 
which  is  probably  best  known  cytologically,  a  brief  account  of  mitosis 
will  be  given  as  described  by  Harper  in  the  ascus  of  Erysipke 
communis.. 


8' 


INTRODUCTION. 


The  ascus  of  this  species  offers  unusually  favorable  material  for  the 
study  of  mitosis  on  account  of  the  clearness  with  which  all  details  are 
brought  out,  and  because  the  three  successive  nuclear  divisions  follow 
each  other  rapidly,  making  it  possible  to  trace  with  unmistakable 
clearness  the  persistence  of  the  centrosome  from  one  nuclear  genera- 
tion  to  the  other.  Since  the  spindles  lie  in  different  planes,  it  is  pos- 
sible also  to  observe,  side  by  side,  the  same  stages  at  different  angles 
in  the  same  field  of  the  microscope.  The  following  refers  especially 
to  the  second  mitosis  in  the  ascus. 


W^V;?.-^' 


Fig.  4. — Mitosis  in  ascus  oi Erysiphe  communis. — (After  Harper.) 

A,  nucleus  in  resting  stage  of  second  nuclear  generation  in  ascus,  the  flattened  or  disk-shaped  centro- 

some closely  applied  to  nuclear  membrane. 

B,  early  prophase  ;  the  kinoplasmic  radiations  have  been  developed  about  the  centrosome. 

C,  D,  E,  F,  successive  steps  in  development  of  spindle. 

G,  mature  spindle,  the  nuclear  membrane  still  persists  at  sides. 

H,  end  of  anaphase ;  connecting  fibers  extend  between  the  daughter  nuclei,  which  are  not  yet  provided 

with  a  nuclear  membrane. 
I,  daughter  nucleus  provided  with  membrane,  kinoplasmic  radiations  present. 
J,  later  stage  in  which  the  polar  radiations  have  disappeared. 

Between  the  successive  nuclear  divisions  in  the  ascus,  the  chromatin 
of  the  daughter  nuclei  does  not  assume  the  complete  resting  condition. 
It  consists  (Fig.  4,  A)  of  an  irregular  net  with  the  angles  of  the 
meshes  somewhat  thickened.  Generally  the  net  lies  tolerably  free  in 
the  nuclear  cavity,  and  a  very  distinct  nucleolus  is  present.  The 
centrosphere  appears  as  a  flattened  disk  closely  applied  to  the  nuclear 
membrane,  giving  the  impression  as  if  the  two  were  grown  together 
(Fig.  4,  A).     The  chromatin  net  appears  also  attached  at  this  place 


NUCLEAR    DIVISION.  9 

and  frequently  forms  a  dense  mass.  These  phenomena  indicate 
clearly  that  chromatin  and  centrosphere  are  in  dii^ect  communication 
through  the  nuclear  membrane.  The  first  step  in  the  division  is 
characterized  by  the  appearance  of  a  well-developed  aster  or  system 
of  radiations  about  the  centrosome.  It  seems  very  probable  here  that 
the  radiations  grow  out  into  the  cytoplasm  from  the  centrosome  as  a 
center.  In  the  development  of  the  radiations  the  nucleus  probably 
cooperates.  At  this  stage  the  chromatin  is  contracted  into  a  dense 
net  toward  the  centrosphere  and  appears  in  close  connection  with  it. 
From  the  chromatin  mass  several  fine  achromatic  threads  extend 
toward  the  nuclear  membrane  (Fig.  4,  B). 

In  the  next  stage  observed,  the  two  poles  of  the  spindle  have  been 
formed,  which  lie  some  distance  apart  on  the  nuclear  membrane 
(Fig.  4,  C).  The  polar  radiations  are  well  developed,  and  from  each 
centrosome  a  cone  of  spindie  fibers  extends  into  the  nuclear  cavity. 
The  diverging  fibers  seem  to  be  inserted  in  the  nuclear  membrane  at 
points  opposite  the  centrosome.  As  in  Dictyota  the  two  systems  of 
fibers  cross  each  other  at  nearly  righ't  angles  without  in  any  way 
uniting.  Whether  the  two  centrospheres  arose  by  a  division  of  the 
primary  centrosphere  cannot  be  stated  with  absolute  certainty,  since 
the  intermediate  stages  between  B  and  C,  Fig.  4,  were  not  observed, 
yet  from  what  is  known  in  Stypocaulon  and  in  Dictyota^  it  seems 
reasonable  to  suppose  that  the  centrosphere  may  undergo  a  division 
in  Erysiphe  also. 

The  chromatin,  at  this  stage,  seems  to  be  reduced  in  mass  to  that 
which  will  appear  in  the  nuclear  plate.  It  lies  distributed  in  irregular 
lumps  among  the  fibers  opposite  the  two  poles.  The  nucleolus  has 
now  disappeared,  or,  in  some  cases,  it  may  remain  in  the  form  of  a 
weakly  staining  residue.  The  spindle  fibers  within  the  nucleus  be- 
come attached  to  the  chromosomes  and  then  contract  strongly,  bringing 
the  chromosomes  into  the  center  of  the  nuclear  cavity  (Fig.  4,  C,  D, 
E,  F).  Some  of  the  fibers  of  the  bent  spindle  appear,  at  this  stage,  to 
extend  uninten-uptedly  from  pole  to  pole.  The  continuous  fibers  are, 
in  all  probability,  formed  by  the  union  of  those  which  are  not  attached 
to  the  chromosomes. 

The  polar  radiations  now  undergo  a  marked  change,  becoming  shorter 
and  thicker,  as  if  drawn  in  toward  the  poles.  The  majority  of  the 
radiations  diverge  only  slightly.  They  are  contracted  into  bundles  or 
brush-like  collections,  which  stand  perpendicular  to  the  surface  of  the 
nucleus.  Some  of  these  radiations,  however,  diverge  somewhat  from 
the  central  group,  but  all  the  polar  radiations  are  not  centered  upon  a 
single  point.     The  pole  of  the  spindle  is  exactly  as  broad  as  the  base  of 


lO  INTRODUCTION. 

the  central  group  of  polar  radiations,  and,  as  will  be  seen  from  Fig.  4, 
E,  F,  G,  the  impression  is  that  the  polar  radiations  and  the  spindle 
contain  the  same  number  of  fibers,  which  are  continued  uninterruptedly 
through  the  poles.  But  the  continuity  of  the  fibers  is  sharply  inter- 
rupted by  an  achromatic  plane  at  the  nuclear  membrane,  through  which 
the  deeply  staining  (violet,  by  the  Flemming  triple  stain)  fibers  pass 
from  nucleus  to  cytoplasm.  Whether  the  spindle  fibers  actually  end 
at  the  nuclear  membrane,  or  whether  their  substance  only  stains  less 
densely  there,  was  not  determined.  However,  the  phenomenon  leaves 
the  impression  that  the  central  body  consists  merely  of  the  bases  of  the 
polar  radiations  closely  crowded  together.  If  the  centrosome  is  an 
individual  organ  here,  it  seems  that  it  must  consist  of  a  very  thin,  flat- 
tened disk,  equal  in  breadth  to  the  blunt  end  of  the  spindle. 

The  poles  of  the  spindle  now  separate  farther  from  each  other, 
whereby  the  spindle  becomes  straight.  The  individual  chromosomes, 
eight  in  number,  which  are  arranged  in  the  equatorial  plate,  are  sharply 
defined,  and  the  nucleus  has  become  somewhat  elongated  (Fig.  4,  G). 
The  polar  radiations  have  again  become  fine  elongated  fibers,  forming 
regular  systems  of  sun-like  radiations. 

As  soon  as  the  daughter  chromosomes  have  reached  the  poles  of  the 
spindle  the  nuclear  membrane  disappears  (Fig.  4,  H).  The  fibers  of 
the  central  spindle  become  now  less  sharply  defined  and  broken  in 
different  places.  Their  number  is  also  gradually  diminished,  their 
substance  soon  being  indistinguishable  from  the  immediately  surround- 
ing cytoplasm.  The  polar  radiations,  however,  form  at  this  stage  a 
more  regular  and  sharply  defined  aster,  owing  to  the  outer  rays  bend- 
ing somewhat  backward  round  the  chromosomes  (Fig.  4,  H).  The 
latter  form  a  dense  mass  in  which  the  individual  elements  are  no 
longer  to  be  distinguished.  The  centrosome  is  likewise  not  to  be 
distinguished  from  the  chromatin  mass  near  which  it  lies.  A  nuclear 
membrane  is  now  formed  about  each  daughter  nucleus,  which  appears 
as  a  small  vesicle  with  the  chromatin  mass  at  the  polar  side  (Fig.  4,  I). 
With  the  further  development  of  the  nuclear  membrane  the  free 
cavity  of  the  nucleus  increases  in  size.  The  chromatin  mass  begins  to 
swell,  and  is  gradually  transformed  into  threads  and  lumps  which  are 
arranged,  at  first,  mostly  along  the  nuclear  membrane,  but  soon 
become  distributed  through  the  nuclear  cavity.  A  nucleolus  now 
appeal's,  and  with  the  further  growth  of  the  nucleus  the  chromatin 
passes  over  into  the  netlike  framework  like  that  in  Fig.  4,  J,  A. 

As  soon  as  the  nuclear  membrane  is  formed,  the  polar  radiations 
begin  to  disappear.  In  Erysiphe  they  seem  to  be  transformed  into  a 
granular  mass  (Fig.  4,   J).     Finally,  when  the  daughter  nucleus  is 


MITOSIS    IN    POLLEN    MOTHKR-CELLS.  II 

mature,  the  centrosphere   remains  as  a   much  flattened   disc  closely 
applied  to  the  nuclear  membrane. 

From  the  foregoing  it  is  clear  that,  although  differing  much  in  detail, 
the  karyokinetic  process  in  Erysiphe  is,  in  general,  similar  to  that  in 
the  brown  algae.  At  our  present  state  of  knowledge,  it  is  difficult  to 
explain  all  the  minor  differences  or  to  form  an  estimate  of  their 
relative  importance. 

MITOSIS  IN  POLLEN  MOTHER-CELLS. 

The  spore  mother-cells  of  certain  Liliacece  and  other  monocotyledo- 
nous  species,  as  well  as  a  few  dicotyledonous  plants  such  as  Helleborus 
and  Podophyllum^  have  become  classical  objects  for  cytological  study, 
and  in  these  genera  the  mitotic  process  is  now  as  well  understood  as  in 
any  other  angiosperms.  The  following  discussion  of  the  first  two 
nuclear  divisions  in  the  spore  mother-cells  of  higher  plants  is  based 
upon  the  author's  own  investigations  made  upon  Lilium  martagon^ 
L.  candidum^  Fritillaria  persica^  Tradescantia  virginica,  Helle- 
borus foetidus  and  Podophyllum  peltatum. 

THE  FIRST  OR  HETEROTYPIC  MITOSIS. 
RESTING  NUCLEUS  AND  DEVELOPMENT  OF  CHROMATIN  SPIREM. 

Soon  after  the  last  nuclear  division  in  the  archesporium,  or  spore- 
bearing  tissue,  which  gives  rise  to  the  pollen  mother-cells,  the  latter 
begin  that  period  of  growth  so  characteristic  of  spore  mother-cells  pre- 
viously to  the  first  mitosis.  The  nucleus  is  relatively  large  with  a 
sharply  defined  membrane,  and  contains  a  fine  linin  network,  in  which 
the  chromatin  granules  are  held,  and  one  or  more  nucleoli.  The 
nucleolus  may  lie  in  a  colorless,  spherical  cavity,  which  seems  sharply 
circumscribed.  The  chromatin  appears  in  larger  and  smaller  granules, 
which  are,  as  a  rule,  regularly  distributed  in  the  linin  thread.  The 
cytoplasm  presents  a  uniform  netlike  structure  (Fig.  5,  A).  This  is 
the  typical  structure  of  a  pollen  mother-cell. 

With  further  growth  of  the  nucleus,  the  chromatin  granules  increase 
in  size,  probably  through  the  union  or  aggregation  of  the  smaller 
granules,  while  at  the  same  time  the  linin  thread  contracts  and  shortens. 
In  this  stage  the  linin  net  consists  of  a  complicated  spirem  or  thread 
with  short  turns.  The  chromatin  granules  have  attained  a  more  uni- 
form size,  and  lie  more  regularly  distributed  in  the  linin  thread  (Fig. 
5,  B).  This  contraction  of  the  linin  thread  and  fusion  of  the  smaller 
chromatin  granules  continues,  so  that  the  nuclear  thread,  which  later 


12 


INTRODUCTION. 


contains  a  row  of  larger  granules  or  disks  (the  Chrovtatinscheiben  of 
the  German  literature)  of  a  tolerably  uniform  size,  becomes  a  hollow 
spirem  whose  irregular  turns  traverse  the  nuclear  cavity  (Fig.  5,  C). 
The  chromatin  disks  have  usually  a  jagged  or  erosed  outline,  which 
shows  that  each  disk  is  composed  of  smaller  granules.  The  chromatin 
disks,  first  carefully  described  by  Strasburger  ('82),  vary  much  among 
themselves  in  size,  and  do  not  always  have  the  same  orientation  in  the 
linin  thread.  This  fact,  together  with  the  twisting  of  the  thread  upon 
its  axis,  which  is  a  mechanical  necessity,  gives  the  impression  of  a 
spirem  composed  of  very  irregular  granules.     This  is  especially  notice- 


FiG.  s. — Pollen  mother-cell  and  early  prophase  of  first  or  heterotypic  mitosis.     A,  F,  Podophyllum 
peltatum.     B-E,  Helleborus  ftetidus . 

A,  typical  pollen  mother-cell,  with  nucleus  in  resting  stage,  and  while  the  cells  are  in  tissue  connection. 

B,  linin  net  with  numerous  small  chromatin  granules. 

C,  spirem  in  which  chromatin  disks  are  of  uniform  size. 

D,  pieces  of  chromatin  spirem  more  highly  magnified;   a,  before  longitudinal  splitting;   ^,  after  longi- 

tudinal splitting. 

E,  the  spirem  has  split  longitudinally;  daughter  segments  show  a  tendency  to  separate. 

F,  the  chromatin  spirem  has  segmented  transversely  into  chromosomes ;   daughter  segments  twisted 

about  each  other.     ( All  figures  represent  sections.) 

able  immediately  after  the  longitudinal  splitting  of  the  chromatin 
granules.  At  this  stage  the  most  careful  staining  is  necessary  to  bring 
out  the  chromatin  disks  clearly,  since  the  linin  retains  the  stain  with 
greater  avidity,  thereby  concealing  the  former.  If  the  nuclear  thread 
be  too  densely  stained,  it  will  appear  more  or  less  homogeneous,  in 
which  case  the  chromatin  disks  manifest  themselves  as  a  succession 
of  enlargements  whose  granular  character  is  concealed.  The  chro- 
matin thread  consists,  therefore,  not  of  a  succession  of  chromatin  disks 


MITOSIS    IN   POLLEN    MOTHER-CELLS.  IJ 

but  of  a  continuous  linin  thread  in  which  are  held  the  chromatin  disks 
or  granules. 

In  an  early  stage  the  nuclear  thread  shows  a  marked  tendency  to  con- 
tract into  a  ball  or  mass  about  the  nucleolus.  The  contraction  into  a 
dense  ball  is  regarded  by  some  observers  as  a  perfectly  normal  occur- 
rence, to  which  the  name  synapsis  has  been  given.  My  own  investiga- 
tions have  convinced  me  that  the  contraction  of  the  nuclear  thread  into 
a  ball  is  in  a  large  measure  due  to  the  reagents,  and  that  synapsis  has 
little  or  no  significance.  It  indicates  probably  a  very  sensitive  con- 
dition of  the  nuclear  thread  or  net  at  the  stage  in  which  the  contraction 
occurs. 

Soon  after  the  nuclear  net  has  developed  into  the  spirem,  as  men- 
tioned, the  chromatin  and  linin  elements  split  longitudinally  (Fig.  5, 
D,  a,  3,  E).  The  daughter  spirems  remain  either  closely  applied  to 
each  other,  or,  as  sometimes  happens,  they  may  separate  for  longer  or 
shorter  intervals.  They  are  always  twisted  upon  each  other,  and,  as 
a  consequence,  the  two  parallel  rows  of  disks  are  not  easily  seen, 
especially  where  the  chromatin  thread  makes  short  turns.  The  twist- 
ing of  the  daughter  spirems  upon  each  other  persists  after  the  trans- 
verse segmentation  of  the  spirem  into  chromosomes,  and  in  very  many 
cases  it  is  still  to  be  seen  during  metakinesis  (Figs.  6,  7). 

Very  frequently  portions  of  the  spirem  which  run  parallel  with 
each  other  are  connected  by  very  fine  threads,  and,  in  some  cases,  as 
in  the  pollen  mother-cells  of  Podophyllum^  very  delicate  cytoplasmic 
threads  seem  to  penetrate  the  nuclear  membrane  and  fasten  themselves 
to  the  chromatin  spirem.  At  this  stage  also  one  or  more  nucleoli,  of 
varying  sizes  and  with  a  homogeneous  or  vacuolate  structure,  are  pres- 
ent. The  nuclear  membrane,  especially  in  Podophyllum^  does  not 
present  from  now  on  the  sharp  contour  of  the  resting  nucleus.  It  seems 
to  consist  merely  of  a  cytoplasmic  boundary  (Fig.  5,  F),  and  as  will 
be  pointed  out  in  a  later  paragraph,  we  may  conclude  that  the  nuclear 
membrane  consists  of  an  extremely  delicate  kinoplasmic  network, 
whose  meshes  in  the  resting  nucleus  are  so  closely  arranged  that  only 
a  sharp  line  is  seen  when  observed  in  optical  section.  As  soon, 
however,  as  the  meshes  widen  with  the  increase  in  size  of  the  nucleus 
the  nuclear  membrane  loses  its  sharp  contour.  It  cannot  be  asserted 
with  absolute  certainty  that  the  fine  threads  extending  from  the  nuclear 
membrane  to  the  chromatin  thread  penetrate  the  membrane  and  con- 
tinue into  the  cytoplasm,  but  in  Podophyllum  the  evidence  seems  to 
be  in  favor  of  such  a  view.  At  any  rate  there  seems  to  be  an  intimate 
connection  maintained  between  chromatin  and  cytoplasm. 

As  karyokinesis  progresses,  the  chromatin  thread  contracts,  becom- 


H 


INTRODUCTION. 


ing  shorter  and  thicker,  and  frequently  no  trace  of  the  longitudinal 
splitting  can  be  recognized.     There  is  thus  formed  the  loose,  hollow 


Fig.  6. — Prophase  and  early  stages  in  development  of  spindle  in^  heterotypic  mitosis  of  pollen  mother 
cell.    A,  B,  Liliunt  candidum.     C,  D,  L.  ■martagon. 

A,  the  kinoplasmic  spindle  fibers  arranged  radially  about  the  nucleus,  large  nucleolus  present,  and  thi 

chromosomes,  each  consisting  of  two  rather  thick  segments  twisted  about  each  other,  lie  along  th< 
nuclear  membrane  or  scattered  through  nuclear  cavity. 

B,  same  developmental  stage  as  A ;   here  the  kinoplasmic  fibers  are  disposed  partly  radially  and  parti] 

in  form  of  a  weft  lying  in  cytoplasm  midway  between  nucleus  and  cell-wall. 

C,  the  spindle  fibers  are  encroaching  upon  the  nucleus,  forming  a  weft  about  it;  the  nuclear  membram 

as  such  has  nearly  disappeared  ;  it  seems  to  have  been  converted  into  fibers. 

D,  multipolar  spindle  complex,  in  which  the  chromosomes  are  irregularly  distributed. 

spirem,    which   segments   by   transverse   division   into   the    chromo- 
somes. 


MITOSIS    IN    POLLEN    MOTHER-CELLS.  1 5 

We  shall  now  leave  the  chromosomes  for  the  present  and  pass  to 
the  development  of  the  spindle. 

DEVELOPMENT  OF  THE  SPINDLE. 

The  development  of  the  spindle  in  pollen  mother-cells  varies  some- 
what in  detail  in  different  plants,  but  it  can  usually  be  referred  to  one 
type.  In  all  cases,  so  far  as  known,  it  arises  as  a  multipolar  structure. 
As  soon  as  the  spirem  is  segmented  into  chromosomes,  and  some- 
times earlier,  the  kinoplasmic  fibers  make  their  appearance  in  the  cyto- 
plasm. The  arrangement  of  the  kinoplasmic  fibers  is  not  quite  the 
same  in  all  cells  of  the  same  anther.  They  may  be  disposed  at  first 
radially  about  the  nucleus  (Fig.  6,  A),  or,  as  in  many  cases,  may  form 
a  weft  about  the  nucleus  midway  between  nuclear  membrane  and  cell- 
wall  (Fig.  6,  B).  The  remaining  cytoplasm  consists  of  a  fibrillar 
structure.  In  this  stage  the  nucleus  is  filled  with  a  fluid  which  does 
not  stain,  namely,  the  nuclear  sap.  The  chromosomes  are  connected 
with  each  other  and  with  the  nuclear  membrane  by  means  of  fine 
fibers,  and  one  or  more  nucleoli  are  present.  The  nucleolus,  how- 
ever, begins  to  break  up  at  this  time,  so  that  one  large  and  several 
smaller  ones  may  be  present. 

The  next  step  in  the  development  of  the  spindle  may  differ  slightly 
in  different  cells,  owing  to  the  orientation  of  the  kinoplasmic  fibers. 
In  those  cells  in  which  these  fibers  are  disposed  radially  about  the 
nucleus,  the  tendency  to  form  poles  manifests  itself  before  the  disap- 
pearance of  the  nuclear  membrane.  Groups  of  radiations  converge 
toward  various  points  near  the  plasma  membrane,  while  others  form  a 
weft  about  the  nucleus  (Fig.  6,  C).  A  little  later  the  nuclear  mem- 
brane is  replaced  by  this  weft,  and  the  fibers  begin  to  enter  the  nuclear 
cavity.  In  some  cases  well-defined  poles  (or  only  a  few)  are  not  as 
yet  present.  In  other  cases  a  greater  number  of  poles  are  formed,  and 
we  have  then  a  very  remarkable  multipolar  complex  of  kinoplasmic 
fibers  surrounding  the  nucleus,  into  wrhich  the  fibers  penetrate  from 
all  sides  (Fig.  7,  E). 

Gradually  more  kinoplasmic  fibers  enter  the  nuclear  cavity  until  it 
can  no  longer  be  recognized  as  such  (Fig.  6,  D).  In  this  complex  of 
spindle  fibers  the  chromosomes  are  irregularly  distributed.  They  are, 
however,  soon  collected  together,  and  to  each  a  bundle  of  fibers  be- 
comes attached.  The  chromosomes  seem  to  be  aggregated  more  closely 
together  by  a  pushing  and  pulling  of  the  spindle  fibei-s.  Owing  to  the 
irregular  arrangement  of  the  chromosomes  and  the  complexity  of  .the 
mass  of  spindle  fibers,  it  is  not  always  possible  to  determine  at  this 
stage  the  exact  manner  in  which  the  fibers  are  fastened  to  the  chro- 
mosomes (Fig.  7,  F). 


i6 


INTRODUCTION. 


The  bipolarity  of  the  multipolar  spindle  now  gradually  manifests 
itself,  and  the  multipolar  structure  rapidly  becomes  a  typical  bipolar 
spindle  in  which  the  chromosomes  are  arranged  in  the  equatorial  plate. 


Fig.  7. — Heterotypic  mitosis  in  pollen  mother-cell  (L,  fHartagan).     Development  of  spindle  continued. 

E,  the  weft  of  spindle  fibers  forms  a  multipolar  complex. 

F,  a  multipolar  complex  in  which  bipolarity  has  begun  to  manifest  itself;  the  weaker  poles  seem  to  be 

drawn  in  or  together. 

G,  bipolarity  is  established  and  chromosomes  more  regularly  arranged  in  equator. 

H,  mature  spindle,  showing  only  3  of  the  12  chromosomes;  chromosomes  fastened  endwise  to  spindle. 

This  transformation  is  probably  brought  about  by  certain  of  the  larger 
poles  converging  toward  a  common  area  or  point,  while  others  are 
drawn  in  (Fig.  7,  G).  The  mature  spindle  is  either  truncated  at  the 
poles  (sometimes  broadly  so)  or  pointed,  and  the  chromosomes  are 


MITOSIS    IN    POLLEN    MOTHER-CELLS.  1 7 

quite  regularly  arranged  in  the  equatorial  plate.  They  are  usually 
radially  disposed,  standing  at  right  angles  to  the  axis  of  the  spindle 
(Fig.  7,  H).  The  spindle  fibers  present  the  following  arrangement: 
to  each  chromosome  are  attached  two  bundles  of  fibers  (one  to  each 
daughter  segment)  which  extend  to  the  poles ;  other  fibers,  the  central 
spindle  fibers,  run  uninterruptedly  from  pole  to  pole,  and  still  others 
diverge  from  the  poles  toward  the  cell  periphery.  This  arrangement 
is  commonly  found  in  all  cells  of  the  higher  plants,  whether  they  be 
reproductive  or  vegetative.  The  spindle  does  not,  as  may  appear  at  the 
first  glance,  present  a  system  of  meridional  fibers  converging  toward 
the  poles,  but,  as  is  easily  seen  from  thin  sections,  the  fibers  cross  and 
anastomose,  giving  the  impression  that  the  spindle  consists  of  a  weft  or 
complex  of  fibers  drawn  out  in  the  direction  of  the  poles,  which,  indeed, 
it  really  is. 

In  spore  mother-cells  of  plants,  the  spindle  fibers  seem  to  be  gener- 
ally of  cytoplasmic  origin,  i.  c,  they  appear  first  in  the  cytoplasm, 
forming  a  weft  about  the  nucleus  or  radiating  from  it.  In  the 
generative  cell  of  gymnosperms  and  in  the  first  division  following 
fecundation  in  these  plants,  it  seems  that  the  fibers  or  many  of  them 
arise  from  kinoplasm,  which  is  in  the  nucleus  or  which  entered  the 
same  in  another  form. 

CHROMOSOMES. 

As  is  well  known,  the  chromatin  spirem,  which  has  split  longitudi- 
nally in  the  early  prophase,  segments  by  transverse  division  into  twelve 
chromosomes,  the  reduced  number,  or  half  the  number  in  the  vegeta- 
tive cells  of  the  sporophyte.  Each  chromosome  consists,  therefore, 
of  two  daughter  segments,  or  daughter  chromosomes,  which  are 
almost  always  twisted  upon  each  other  (Fig.  7,  H  ;  Fig.  8).  After 
the  segmentation  of  the  spirem  into  chromosomes,  these  contract, 
thereby  becoming  shorter  and  thicker.  Previous  to  the  disappear- 
ance of  the  nuclear  membrane,  they  lie  near  it  or  are  scattered 
throughout  the  nuclear  cavity  (Fig.  6,  B).  In  Lilium^  the  daughter 
chromosomes  are,  as  a  rule,  closely  applied  to  each  other,  but  in 
many  cases  they  tend  to  become  separated  soon  after  segmentation,  so 
that  various  forms  of  chromosomes  result,  such  as  rings,  loops,  X-  and 
V-shaped  forms,  depending  upon  the  manner  in  which  the  daughter 
segments  are  oriented  toward  each  other  (Fig.  8,  A  to  K).  These 
various  forms  persist  and  may  be  found  in  the  nuclear  plate  of  the 
mature  spindle. 

The  following  will  explain  the  manner  in  which  the  more  fre- 
quently occurring  forms  are  brought  about  in  Lilium^  Podophyllum 
and  in  many  other  higher  plants : 


i8 


INTRODUCTION. 


The  daughter  segments  often  diverge  at  one  or  at  both  ends  (Fig.  8, 
B,  C).  In  other  cases  they  may  be  bent  and  in  contact  only  near  the 
middle  (Fig.  8,  D).  If  the  daughter  segments  adhere  at  the  ends, 
and  bend  away  from  each  other  near  the  middle,  a  ring  results 
(Fig.  8,  E).  Ring-shaped  chromosomes  may  be  so  bent  as  to  bring 
the  opposite  ends  near  each  other,  in  which  case  we  have  a  ring 
partly  folded  upon  itself.  This  is  true  in  a  measure  in  Fig.  8,  E. 
When  the  segments  forming  a  ring  separate  slightly  at  one  end,  an 
open  ring  is  produced. 

A  Y-shaped  chromosome  will  result  when  the  segments  are  con- 
tiguous for  a  part  of  their  length  but  diverge  at  one  end  (Fig.  8,  F). 
Sometimes  the  daughter  segments  adhere  near  the  middle  but  diverge 


H    ■  I  J         K 

Fig.  8. — Heterotypic  mitosis  {Liltum  martagon).    Diflferent  forms  of  chromosomes. 
A,  B,  C,  D,  chromosomes  from  prophase.     E-K,  from  equatorial  place. 
E,  ring-shaped,  F,  Y-shaped,  and  J,  typical  X-shaped  chromosomes. 
G,  H,  1,  and  K,  other  forms  commonly  met  with  in  Lilium. 

at  both  ends,  so  that  they  may  be  crossed ;  this  gives  rise  to  the  X- 
shaped  chromosome  (Fig.  8,  J).  Instances  are  also  met  with  in  which 
the  segments  of  the  X-shaped  chromosome  fuse  completely  at  one  end, 
and  the  chromosome  appears  as  a  continuous  rod,  folded  in  such  a  man- 
ner that  the  opposite  ends  are  brought  together.  In  this  way  loops  and 
incomplete  rings  are  produced  (Fig.  8,  K).  In  Fig,  8,  G,  H,  and  I 
are  forms  of  chromosomes  that  are  of  frequent  occurrence.  The  orien- 
tation of  the  daughter  segments  toward  each  other,  which  results  in  the 
different  forms  of  chromosomes  described,  is,  in  all  probability,  of  no 
special  importance,  since  two  or  more  of  these  forms  may  be  seen  in 
the  same  nucleus. 

In  Tradescantia^  between  the  time  of  the  segmentation  of  the  spirem 
into  chromosomes  and  the  mature  spindle,  the  daughter  segments  often 
contract  into  the  form  of  short,  thick  crescents.     These  may  adhere  at 


MITOSIS    IN    POLLEN    MOTHER-CELLS.  I9 

the  points  of  the  crescents  to  form  ring-like  chromosomes  (Fig.  9,  D, 
at  the  right).  In  the  majority  of  cases,  however,  they  adhere  at  only 
one  end,  and  under  such  circumstances  each  chromosome  consists  of 
two  thick  and  slightly  curved  pieces  placed  end  to  end,  and  as  they 
are  oriented  tangentially  upon  the  spindle,  reach  nearly  from  pole  to 
pole  (Fig.  9,  D). 

The  chromosomes  in  Podophyllum  present  the  same  variety  of  forms 
found  in  Lilium  and  Tradescantia.  Here  the  segments  may  be  in 
close  contact,  side  by  side,  or  form  loops,  rings,  X's,  and  Y's.  Per- 
haps the  majority  of  chromosomes  in  Podophyllum  present  the  form 
last  mentioned  for  Tradescantia. 

In  Lilium  the  chromosomes,  vs^hen  in  the  nuclear  plate,  are  usually 
arranged  with  much  regularity  about  the  periphery  of  the  spindle. 
The  majority  are  fastened  to  the  fibers  at  the  ends,  and  stand  radially 
to  the  axis  of  the  spindle  (Fig.  7,  H).  When  observed  from  the  pole 
in  this  stage,  they  are  seen  to  radiate  like  the  spokes  of  a  wheel  from 
the  central  spindle  fibers.  But  all  the  chromosomes  are  not  so  regu- 
larly oriented  upon  the  spindle,  and  their  manner  of  attachment  to  the 
fibers  is  also  variable.  As  will  be  seen  in  Fig.  8,  F-K,  they  may  be 
fastened  to  the  spindle  at  some  distance  from  one  end  or  near  the  mid- 
dle. Those  that  are  quite  regularly  ring-shaped  are  attached  near  the 
middle  of  each  segment.  In  all  these  cases,  the  chromosomes  are 
placed  tangentially  upon  the  spindle.  The  X-,  Y-,  and  loop-shaped 
chromosomes  are  usually  fastened  to  the  spindle  as  indicated  in  Fig. 
8,  F,  J,  K.  Karyokinetic  figures  are  not  rare  in  which  two  or  more 
of  the  different  forms  of  chromosomes,  with  their  different  orientations 
and  different  methods  of  attachment  to  the  fibers,  are  found  in  the 
same  spindle.' 

The  stage  of  the  mature  spindle  persists  some  time  and  evidently 


•  Other  interpretations  of  the  chromosomes  appearing  in  the  first  mitosis  have  been  given  by  different 
observers  and  by  the  same  investigator  at  different  times,  owing  to  the  trend  of  theoretical  considerations. 
One  of  these,  which  was  announced  as  early  as  1884  by  Heuser  for  Tradescantia  virginica  (Beobach- 
tung  uber  Zellkerntheilung.  Bot.  Centralblt.,  17  :  1884)  and  which  has  very  recently  received  support  by 
Strasburger  and  others  (Ueber  Reduktionstheilung.  Sitzbr.  der  Konig.  Preuss.  Akad.  der  Wiss.,  18  : 
t-28,  1904)  is  that  the  two  segments  of  each  chromosome  appearing  in  the  equatorial  plate  of  the  first 
mitosis  are  not  the  result  of  the  longitudinal  splitting  of  the  spirem  occurring  in  the  early  prophase,  but 
are  formed  by  the  folding  together  or  approximation  of  two  chromosomes,  each  consisting  of  the  two 
daughter  segments  resulting  from  the  longitudinal  splitting.  Each  chromosome  is  therefore  a  bivalent 
chromosome,  and  the  first  or  heterotypic  mitosis  is  a  qualitative  cr  reducing  division,  whereas  the  second 
mitosis  is  equational,  the  segments  separating  along  the  line  of  the  longitudinal  split.  Strasburger  bases 
his  conclusion  mainly  upon  data  obtained  from  studies  of  the  pollen  mother-cells  of  Galtonia  candicaus. 
The  figures  which  he  gives  in  support  of  this  view  in  the  paper  cited  seem  to  me  to  be  far  from  convinc- 
ing. Moreover,  Jules  Berghs,  in  a  recent  study  of  the  prophase  of  the  heterotypic  mitosis  in  Allium 
fistulosunt  and  Lilium  lanci/olium  (j/tfc/Vjjxwi)  (La  Cellule,  21:  173-188, 1904), shows  clearly,  in  a  careful 
series  of  stages,  that  the  two  segments  of  each  chromosome  are  the  result  of  the  longitudinal  fission  and 
not  that  of  a  folding  together  or  approximation  of  two  chromosomes.  Unfortunately  the  papers  cited 
reach  me  too  late  for  further  consideration,  as  these  pages  are  already  in  press. 


20  INTRODUCTION. 

represents  a  slight  pause  in  the  process  of  mitosis.     For  this  reason  it 
is  the  stage  most  easily  obtained  and  most  frequently  observed. 

METAKINESIS. 

Up  to  the  stage  of  the  mature  spindle,  as  in  Fig.  7,  H,  each 
chromosome  is  seen  to  consist  of  two  daughter  segments  oriented  in 
one  of  the  ways  described  above.  As  soon,  however,  as  these  seg- 
ments begin  to  separate  in  metakinesis,  each  splits  longitudinally  in  a 
plane  at  right  angles  to  the  longitudinal  splitting  which  took  place  in 
the  prophase.  In  some  instances,  and  when  the  chromosomes  are 
viewed  from  the  end,  each  is  seen  to  be  composed  of  four  rods,  the 
four  granddaughter  segments,  placed  side  by  side  in  pairs,  forming  a 
tetrad.  Fig.  9,  A.  As  a  rule  the  granddaughter  segments  cannot  be 
definitely  recognized  until  the  daughter  segments  have  separated 
somewhat.  Having  almost  or  quite  separated,  the  daughter  segments 
are  seen  to  be  in  the  form  of  a  V,  although  it  never  should  be  for- 
gotten that  V's  do  not  invariably  result.  As  the  result  of  the  second 
longitudinal  splitting,  each  typical  V-shaped  daughter  chromosome 
consists  of  two  granddaughter  segments  which  adhere  or  are  even 
fused  at  the  ends  to  which  the  spindle  fibers  are  fastened,  while  the 
opposite  ends  diverge  (Fig.  9,  B).  It  frequently  happens  that  the 
opposite  ends  of  the  granddaughter  segments  do  not  diverge,  but  lie 
more  or  less  in  contact  side  by  side,  so  that  the  retreating  daughter 
chromosomes  consist  of  two  applied  rods  (Fig.  9,  F,  the  middle  pairs) . 
In  some  cases,  as  already  mentioned,  the  ends  of  the  granddaughter 
segments  forming  the  angle  of  the  V  fuse,  so  that  the  V  appears  to  be 
one  piece  formed  by  bending.  The  bent  or  contorted  condition  of  the 
granddaughter  segments  during  metakinesis  is  due  to  the  previous 
twisting  of  the  daughter  chromosomes  upon  each  other. 

If  the  chromosomes  be  in  the  form  of  rings,  as  shown  in  Fig.  8,  E, 
it  is  evident  that  the  separating  daughter  chromosomes  may  also  be  in 
the  form  of  a  V  or  U,  but  such  V's  and  U's  will  be  produced  by  a 
bending  of  the  daughter  segments.  This  is  true  in  a  great  many  cases 
in  Lilium  and  in  other  plants,  among  both  monocotyledonous  and 
dicotyledonous  species.  In  such  cases  each  U  or  V  is  invariably 
double,  as  the  result  of  the  second  longitudinal  fission — that  is,  the 
granddaughter  segments  are  U-shaped  and  closely  applied  to  each 
other  (Fig.  9,  F,  right  and  left).  Sometimes  these  granddaughter  seg- 
ments may  separate  slightly,  giving  the  impression  of  two  similar 
daughter  chromosomes  lying  one  just  beneath  the  other.  This  is  one 
of  the  several  phenomena  that  have  led  to  erroneous  interpretations 
of  the  chromosomes. 


MITOSIS    IN    POLLEN    MOTHER-CELLS. 


21 


In  Fig.  9,  C,  on  the  left,  is  shown  a  chromosome  in  metakinesis, 
which  is  fastened  to  the  spindle  near  the  middle.  Each  daughter  seg- 
ment, which  is  split  longitudinally,  is  in  the  form  of  a  U-like  figure, 


Fig.  9. — Heterotypic  mitosis.     Meta-  and  anaphases.     A,  B,  C,  and  F,  Lilium.     D,  Iradtscantia. 

E,  Podophyllum. 

A,  metakinesis  beginning ;  viewed  from  the  end,  each  chromosome  is  seen  to  consist  of  four  rods,  due 

to  the  second  longitudinal  splitting,  which  has  taken  place  at  right  angles  to  the  first. 

B,  metakinesis  accomplished ;  ends  of  granddaughter  chromosomes,  which  are  directed  toward  equator, 
__      diverge,  giving  rise  to  the  well-known  V-shaped  elements;  in  B  all  chromosomes  arc  fastened  to 

spindle  fibers  at  the  ends. 

C,  chromosome  on  left  was  in  form  of  an  incomplete  ring ;  segments  fastened  at  place  of  bending ;  in  this 

case  the  U-  or  V-shaped  elements  owe  their  form  to  a  bending ;   the  chromosome  on  the  right  was 
attached  endwise. 

D,  mature  spindle  of  Tradescantia.     E,  F,  anaphase ;  the  retreating  pairs  of  granddaughter  segments 

are  rods  hooked  at  one  end,  or  U's. 

in  which  one  limb  seems  a  little  longer  than  the  other.  This  chromo- 
some may  originally  have  been  a  complete  ring,  as  in  Fig.  8,  E,  in 
which  the  segments  had  separated  at  one  end  in  advance  of  the  other, 


ii  INTRODUCTION. 

or  it  may  have  had  this  foinii  at  an  earlier  stage.  The  chromosome  at 
the  right  in  this  figure  (Fig.  9,  C),  was  attached  to  the  spindle  end- 
wise, and  the  retreating  granddaughter  segments  will  probably  form 
Vs.  If  the  chromosome  on  the  left  were  rotated  45°,  so  that  the  seg- 
ments would  be  seen  in  profile,  we  might  have  the  picture  of  two 
double  V's  or  U's  about  to  separate,  for,  as  shown  in  the  figure,  the 
free  ends  of  the  pairs  of  granddaughter  elements  tend  sometimes  to 
diverge.  The  two  chromosomes  in  this  figure,  which  belong  to  the 
same  spindle,  show  clearly  how  figures  of  the  same  shape  may  be  pro- 
duced in  different  ways.  In  the  one  on  the  right  the  chromosome  was 
probably  attached  to  the  spindle  by  the  end,  and  the  V's  are  formed  by 
the  divergence  of  the  free  ends,  while  that  on  the  left  was  fastened  near 
the  middle  of  each  segment,  and  the  V-  or  U -shape  of  the  retreating 
segments  is  the  result  of  a  bending. 

In  such  chromosomes  as  Fig.  8,  G,  H,  I,  the  retreating  elements 
may  retain  their  present  form,  or  they  may  be  bent  during  metakinesis 
into  U's  or  V's.  When  the  daughter  segments  of  such  chromosomes 
are  separated,  they  must  untwist,  and  it  is  reasonable  to  suppose  that 
the  force  necessary  to  separate  them  when  twisted  will  be  sufficient  to 
bend  the  segments  into  a  U-  or  V-like  figure. 

THE  ANAPHASE. 

The  pairs  of  granddaughter  segments,  as  they  pass  toward  the  poles, 
are  in  the  form  of  contiguous,  straight,  or  undulating  rods,  V's  or  U's, 
or,  in  case  one  limb  of  the  last  two  named  figures  be  much  longer 
than  the  other,  as  is  sometimes  observed,  the  retreating  elements  will 
be  in  the  form  of  hooks.  Even  in  those  cases  in  which  both  grand- 
daughter segments  are  nearly  straight  or  undulating  rods  of  equal 
length,  each  is  often  slightly  bent  or  hooked  at  the  end  fastened  to  the 
spindle  fibers,  or  the  segments  may  be  bent  at  both  ends. 

The  daughter  chromosomes  in  Podophyllu?n  and  Tradescantia 
show  with  great  clearness  their  double  character  during  the  anaphase 
(Fig.  9,  E).  The  granddaughter  segments  generally  lie  close  side  by 
side,  although  cases  in  which  they  are  slightly  separated  are  now  and 
then  to  be  observed.  There  are  in  these  genera  also  variations  in  the 
forms  of  the  chromosomes  which  may  be  explained  in  the  same  man- 
ner as  in  Lilium. 

The  retreating  chi'omosomes  and  the  structure  of  the  spindle  suggest 
that  the  segments  are  conveyed  to  the  poles  by  a  pushing  and  pulling 
action  of  the  spindle  fibers. 


MITOSIS    IN    POLLEN    MOTHER-CELLS.  2^ 

THE  TELOPHASE. 

As  soon  as  the  daughter  chromosomes  arrive  at  the  poles,  they 
approach  each  other  very  closely,  so  that,  in  many  cases,  the  separate 
individuals  cannot  be  recognized.  But  very  frequently  the  segments 
do  not  become  so  closely  crowded  together,  and  the  manner  in  which 
the  daughter  spirem  is  formed  can  be  followed  with  accuracy.  The 
formation  of  the  spirem  can  best  be  observed  when  the  granddaughter 
segments  arrive  at  the  poles  in  the  form  of  the  familiar  V-shaped 
figures.  Generally  the  ends  forming  the  angles  of  the  V  fuse  first, 
unless  this  has  already  been  accomplished  ;  then  the  free  ends  meet  end 
to  end  and  unite  (Fig.  lO,  G).  In  this  way  there  is  formed  a  continuous 
single  spirem  in  which  the  identity  of  the  individual  segments  or 
granddaughter  chromosomes  is  lost. 

If  all  the  daughter  chromosomes  were  regularly  V-  or  U-shaped  the 
spirem  would  be  regular,  consisting  of  an  orderly  series  of  nearly 
uniform  turns ;  but  the  spirem  rarely  shows  such  regularity,  because 
the  chromosomes  vary  in  size  and  shape  and  in  the  manner  in  which 
the  granddaughter  segments  are  oriented  with  respect  to  each  other 
in  the  several  pairs.  During  the  reconstruction  of  the  daughter 
nucleus,  the  chromosomes  tend  to  reticulate,  that  is,  to  become 
irregular  and  lumpy,  so  that  an  irregular  skein  or  net  results.  This 
is  less  pronounced  in  Liliuin  than  in  many  other  plants. 

The  fact  that  pairs  of  granddaughter  segments  arrive  at  the  poles  in 
diffei*ent  forms,  such  as  V's,  double  U's,  and  pairs  of  parallel  rods, 
shows  clearly  that  in  such  cases  the  resulting  spirem  must  be  very  irreg- 
ular. The  chromosomes  are  generally  so  closely  crowded  together 
that  it  is  not  possible  to  determine  with  certainty  just  how  the  variously 
shaped  pairs  of  segments  behave.  But  it  is  reasonable  to  suppose  that 
the  segments  of  the  double  U's  and  those  of  contiguous  rods  must  first 
separate  in  order  to  unite  end  to  end,  for  no  case  has  been  clearly  made 
out  in  Lilium  in  which  a  part  of  the  spirem  is  formed  double. 

The  newly  formed  daughter  spirem  is  close  with  relatively  short 
turns  (Fig.  lo,  G,  H).  Between  each  twa  extends  the  beautiful  system 
of  connecting  fibers,  which  represents  the  central  fibers  of  the  spindle. 
Fibers  are  also  present  which  extend  from  each  spirem  toward  the 
plasma  membrane  in  the  direction  of  the  equator.  Some  of  these 
reach  the  plasma  membrane,  while  others  seem  to  end  blindly  in  the 
cytoplasm,  or  pass  over  into  its  thread-work.  In  Lilhi?n  there  are  no 
polar  radiations. 

The  system  of  connecting  fibers  soon  becomes  barrel-shaped,  and 
the  cell-plate  makes  its  appearance  in  the  equatorial  region.  We 
shall  return  to  the  formation  of  the  cell-plate  beyond. 


24 


INTRODUCTION. 


The  nuclear  membranes  are  not  formed  about  the  daughter  nuclei  In 
Lilium  mart  agon  until  after  the  division  of  the  cell,  at  least  in  many 
instances.  Soon  after  the  division  of  the  cell,  however,  the  nuclear 
membranes  are  laid  down.     In  ail  plants  examined,  each  appears  first 


Fig.  io. — Telophase  and  daughter  nucleus  of  heterotypic  mitosis  (^Lilium  tnartagon). 
G,  daughter  spirem  formed  by  union  of  granddaughter  segments  end  to  end ;  each  daughter  spirem  is  in 

the  form  of  a  disk  from  whose  edges  kinoplasmic  fibers  extend  out  in  direction  of  cell-wall ;  system 

of  connecting  fibers  slightly  bulged  out  at  middle. 
H,  the  cell-plate  appears  in  center  of  system  of  connecting  fibers. 

I,  J,  cell-division  is  completed,  but  the  daughter  nuclei  are  not  yet  provided  with  membranes. 
K,  a  daughter  nucleus  at  a  later  stage  with  nuclear  membrane;  chromatin  spirem  continuous,  the  free 

ends  having  been  made  by  knife  in  sectioning. 

as  a  weft  of  kinoplasmic  fibers,  which  are  undoubtedly  dei'ived  fi-om 
the  spindle.  It  is  interesting  to  note  that  in  Lilium  and  Podophyllum 
the  nuclear  membrane  appears  in  the  same  form  in  which  it  disap- 
peared during  the  formation  of  the  spindle.  The  fact  that  the  nuclear 
membrane  arises  first  as  a  weft  of  kinoplasmic  fibers  is  a  strong  proof 
that  it  is  of  a  kinoplasmic  nature. 


MITOSIS    IN    POLLEN    MOTHER-CELLS.  25 

The  young  weft-like  nuclear  membrane  encloses  a  cavity  containing 
the  chromatin  and  little  or  no  other  staining  material.  With  further 
development  the  kinoplasmic  weft  is  transformed  into  the  typical 
nuclear  membrane,  appearing  in  section  as  a  sharp  line,  and  the 
daughter  spirem  becomes  loose  and  open,  [n  the  mature  daughter 
nucleus  the  spirem  is  continuous  and  of  a  tolerably  uniform  thickness. 
In  some  cases  it  is  rather  regular,  consisting  of  long  turns  arranged  in 
the  form  of  a  wreath  (Fig.  lo,  K),  but  in  the  majority  of  instances 
the  spirem  is  irregular,  with  long  and  short  turns  so  disposed  that  its 
course  cannot  be  easily  followed.  This  condition  of  the  spirem  is  in 
all  .probability  due  to  the  variously  shaped  chromosomes  mentioned 
in  a  preceding  paragraph. 

THE  NUCLEOLUS. 

In  the  resting  nucleus  and  during  the  prophase,  one  or  more  nucle- 
oli are  present.  These  nucleoli  take  on  a  deep  red  or  reddish  purple 
color  with  the  Flemming  triple  stain.  They  sometimes  present  a  uni- 
form structure,  but,  as  a  rule,  the  larger  nucleoli  especially  reveal  one 
or  more  vacuoles.  As  has  been  mentioned  in  a  preceding  paragraph, 
the  nucleolus  very  frequently  lies  within  a  spherical  space  which 
appears  in  optical  section  as  a  colorless  court  about  it.  This  phe- 
nomenon is  especially  striking  in  vegetative  cells  of  higher  plants, 
such  as  in  root  tips  of  Vicia  faba  and  Zea  mays.  Experiments 
seem  to  show  that  the  colorless  space  surrounding  the  nucleolus 
contains  something  more  than  a  mere  watery  fluid  which  is  extracted 
in  dehydration.  By  subjecting  roots  of  Vtcta,  Zea  and  others  to  a 
strong  centrifugal  force,  the  author  (Mottier,  '99)  found  that  the 
nucleolus  together  with  its  surrounding  colorless  court  was  thrown 
out  of  the  nucleus  into  the  cytoplasm.  The  expelled  nucleolus  was 
still  surrounded  by  its  colorless  court — a  fact  that  seems  to  show  that 
the  colorless  substance  has  a  specific  gravity  much  greater  than  other 
constituents  of  the  nucleolus,  and  that  it  may  be  provided  with  its  own 
membrane.  This  colorless  substance  may  represent  unorganized 
nucleolar  matter. 

Frequently  before  the  nuclear  membrane  disappears  a  disorganiza- 
tion begins  by  which  the  nucleolus  is  broken  up  into  several  smaller 
nucleoli  (Fig.  6,  C).  As  the  nuclear  membrane  fades  away,  and  the 
kinoplasmic  fibers  enter  the  nuclear  cavity,  numerous  bodies  are  found 
distributed  in  the  cytoplasm  which  stain  exactly  as  nucleoli,  and  there 
is  no  doubt  that  these  bodies  represent  nucleolar  substance.  These 
extra-nuclear  nucleoli  were  found  to  be  more  abundant  in  Lilium 
viartago7t.     In  Lilium  candidum  there  may  be  none,  or  only  a  few 


36  INTRODUCTION. 

small  ones,  at  corresponding  stages  of  mitosis.  The  piesence  or  ab- 
sence of  extra-nuclear  nucleoli  may  not  depend  so  much  upon  the 
plant,  perhaps,  as  upon  the  condition  or  activity  of  the  cell.  From 
the  spindle  stage  of  the  first  to  the  end  of  the  second  division  there  is 
no  noticeable  regularity  in  the  behavior  of  these  bodies.  In  different 
cells  in  the  same  stage  of  mitosis  they  may  be  present  or  wholly  want- 
ing. Even  after  the  daughter  nuclei  are  provided  with  membranes, 
and  a  nucleolus  is  present  in  each,  extra-nuclear  nucleoli  are  to  be  fre- 
quently seen  in  the  cytoplasm.  The  same  holds  also  for  the  second 
mitosis.  A  careful  investigation  of  the  behavior  of  the  nucleolus  in 
both  Thallophyta  and  higher  plants  has  shown  that  the  nucleolus 
appearing  in  the  daugliter  nucleus  is  not  one  of  the  extra-nuclear 
nucleoli  which  happened  to  lie  near  the  chromatin,  or  in  such  a  posi- 
tion as  to  be  included  by  the  nuclear  membrane,  but  that  the  nucleolus 
arises  anew  in  each  daughter  nucleus.  The  nucleolus  appearing  in 
the  daughter  nucleus  arises  usually  near  or  in  contact  with  the  chro- 
matin thread,  but  it  is  not  implied  that  the  nucleolus  represents  reserve 
chromatin. 

In  the  higher  plants  and  in  those  with  typical  nuclei  the  morpho- 
logical evidence  furnished  by  a  study  of  karyokinesis,  as  well  as  the 
evidence  of  experimental  physiology,  goes  to  show  that  the  nucleolus 
in  such  plant  cells  lepresents  so  much  food  material  which  can  be 
drawn  upon  by  the  cell  according  to  its  needs.  Whenever  the  activity 
of  the  cell  is  more  intense,  the  nucleolar  substance  tends  to  become 
diminished,  and  it  matters  not  whether  the  activity  is  directed  toward 
constructive  work  or  the  production  of  energy.  It  is  true  that  in  some 
cases  the  food  material  furnished  by  the  nucleolus  seems  to  be  used  in 
a  large  measure  by  the  chromatin,  for  example,  in  Dictyota^  but  in 
others  by  other  parts  of  the  living  substance,  as  in  the  growth  of  the 
spindle  or  cell  plate.  In  certain  species  of  Spirogyra  (Wisselingh, 
'98),  in  which,  as  it  has  been  claimed  by  several  investigators,  the 
nucleolus  furnishes  directly  one  or  more  chromosomes,  greater  diffi- 
culties present  themselves.  It  is  not  improbable  that  the  nucleolus  of 
such  plants  as  Spirogyra  may  possess  a  totally  different  composition 
from  that  of  the  typical  nucleolus,  and  we  may,  therefore,  speak  with 
propriety  of  chromatin  nucleoli.  However  the  behavior  of  the 
nucleolus  is  not  well  enough  known  in  the  plant  kingdom  to  justify 
any  attempt  to  harmonize  all  the  facts  now  known.  Applied  to  the 
higher  plants  the  above  conclusion  seems  to  be  very  reasonable,  since 
the  facts  there  are  almost  wholly  confirmatory. 


MITOSIS    IN    POLLEN    MOTHKR-CKLLS.  2*J 

THE  SECOND  OR  HOMOTYPIC  MITOSIS. 

In  the  pollen  mother-cell  of  Lilium^  the  daughter  nucleus  does  not 
pass  into  the  complete  resting  stage,  although  in  some  cases  the 
chromatin  tends  to  become  reticulated.  In  the  homologous  division 
in  the  embryo-sac,  the  daughter  nucleus,  on  the  contrary,  passes  into 
a  structure  which  approaches  closely  that  of  the  resting  condition.  In 
Tradescantia  the  chromatin  of  the  daughter  nucleus  reticulates  more 
than  in  Lilium  while  in  certain  dicotyledonous  species,  e.  g.^  Lirio- 
dendren  and  Magnolia  (Andrews,  'oi),  a  complete  resting  condition  is 
reached. 

The  spindle  in  Lilium  and  in  all  other  plants  investigated  by  the 
author  arises  also  as  a  multipolar  complex  of  fibers.  The  develop- 
ment of  the  multipolar  structure  and  its  transformation  into  the  typical 
bipolar  spindle  differ  in  no  essential  from  that  already  described  for 
the  first  mitosis. 

In  Lilium^  it  is  very  evident  that  the  spirem  does  not  segment 
completely  into  chromosomes  before  the  disappearance  of  the  nuclear 
membrane.  The  spirem  does  not  split  longitudinally  in  this  division, 
since  that  part  of  the  process  was  accomplished  in  the  preceding 
mitosis,  but  during  the  transformation  of  the  multipolar  into  the 
bipolar  spindle  the  chromatin  skein  segments  into  the  chromosomes, 
which  are  arranged  in  pairs  in  the  nuclear  plate. 

Within  the  complex  of  spindle  fibers,  the  spirem,  or  pieces  of  it, 
provided  it  has  partly  segmented,  are  somewhat  crowded  together. 
The  various  turns  are  greatly  entangled,  kinked  and  knotted,  so  that 
the  segments  cannot  be  accurately  traced  out.  In  only  the  most 
favorable  cases  at  this  stage  can  a  few  segments  or  parts  of  the 
spirem  be  followed  definitely  throughout  their  entire  length  (Fig.  1 1 ,  A). 
The  kinked  and  entangled  condition  of  the  skein  or  its  segments  is  due 
doubtless  to  the  irregularity  of  the  spirem,  for  were  the  turns  all  of  a 
uniform  shape  and  size  a  less  complicated  arrangement  would  result. 
The  appearance  of  the  chromatin  during  the  development  of  the  spindle 
suggests  that  the  chromosomes  were  brought  to  a  more  regular  arrange- 
ment in  the  nuclear  plate  by  a  pushing  and  pulling  of  the  fibers. 

Judging  fi-om  the  form  of  certain  chromosomes  which  stand  out  by 
themselves,  and  which  can  be  traced  throughout  their  entire  length 
during  the  development  of  the  spindle  or  in  the  nuclear  plate,  it  seems 
that  the  spirem,  or  a  part  of  it  at  least,  segments  into  pieces  compris- 
ing the  two  segments  of  a  chromosome,  i.  e.,  the  two  granddaughter 
chromosomes  of  the  first  division,  and  that  these  pieces  may  correspond 
to  long  turns  or  loops  of  the  spirem  (Fig.  1 1,  B,  C).     These  loops  are 


28 


INTRODUCTION. 


fastened  to  the  fibers  either  at  the  free  ends  or  at  the  place  of  bending. 
Now,  in  order  that  the  two  segments  of  such  a  chromosome  may  come 
into  contact  side  by  side,  as  is  frequently  the  case,  the  parallel  parts  of 
the  loop  need  only  be  brought  closely  together.     This  may  happen 


Fig.  II. — Second,  or  homotypic  mitosis,  in  pollen  mother-cells  {Liliunt). 

A,  multipolar  stage  of  spindle ;  chromatin  spirem  not  completely  segmented  into  chromosomes, 

B,  bipolarity  established;  chromosomes  more  regularly  arranged. 

C,  mature  spindle ;  chromosomes  more  regularly  disposed  in  equator,  placed  radially  or  tangentially 

on  spindle. 
D,  metakinesis.     E,  the  anaphase. 

before  the  spirem  is  completely  segmented,  for  in  many  chromosomes 
the  two  segments  are  very  closely  applied  and  twisted  upon  each  other 
before  the  spindle  is  mature.  In  like  manner  parallel  portions  of  the 
spirem  may  come  in  contact  either  before  or  after  segmentation.  In 
many  other  cases,  both  in  the  mature  spindle  and  during  its  develop- 


MITOSIS    IN    POLLEN    MOTHER-CELLS.  29 

ment,  the  two  segments  are  separated  from  each  other,  being  in  con- 
tact only  at  the  ends  which  are  attached  to  the  spindle  fibers.  Under 
this  circumstance  one  segment  may  lie  tangentially  on  one  side  of  the 
equator  and  the  other  on  the  other.  Otlier  instances  are  observed  also 
in  which  the  two  segments  may  lie  parallel  in  pairs,  but  not  in  contact 
when  arranged  in  the  nuclear  plate  or  at  an  earlier  stage.  Such  cases 
as  the  two  last  mentioned  would  seem  to  indicate  that  the  spirem,  or  a 
part  of  it,  is  segmented  into  the  granddaughter  chromosomes,  and  that 
these  are  then  brought  together  in  pairs.  It  is  also  probable  that  pieces 
of  the  segmented  spirem,  which  are  nearly  straight,  or  only  a  little 
curved,  may  consist  of  two  granddaughter  segments,  and  these  are 
brought  side  by  side  by  the  folding  of  the  piece  at  or  near  the  middle, 
so  that  the  free  ends  are  brought  into  apposition,  after  which  the 
piece  is  severed  at  the  point  of  bending.  From  a  careful  study  of  the 
second  mitosis  in  the  pollen  mother-cells  of  Lilium^  Podophyllum^ 
Tradescantia  and  others,  the  author  is  inclined  to  believe  that  the 
spirem  may  segment  in  the  different  ways  just  mentioned.  However, 
the  daughter  spirem  segments  transversely  into  the  granddaughter 
chromosomes,  and  during  the  development  of  the  spindle  these  are 
arranged  more  or  less  in  pairs  in  the  nuclear  plate  (Fig.  ii,  C). 

In  the  nuclear  plate,  the  chromosomes  are  oriented  either  radially, 
obliquely,  or  tangentially  to  the  major  axis  of  the  spindle.  The 
segments  may  be  straight  or  variously  bent,  and,  in  either  case,  fre- 
quently twisted  upon  each  other.  In  Lilium^  the  segments  are 
frequently,  perhaps  in  the  majority  of  cases,  variously  twisted,  kinked 
or  knotted,  so  that  they  can  be  followed  for  only  a  part  of  their 
length.  In  many  cases,  the  kinked  and  twisted  chromosomes  seem 
to  be  so  contracted  as  to  form  lumps.  This  is  true  also  in  Trade- 
scantia and  in  numerous  other  plants.  The  bent,  kinked,  and 
twisted  condition  of  the  chromosomes  seems  to  be  due  to  the  irregu- 
larity of  the  spirem,  for  it  seems  probable  that,  were  all  the  turns  of 
the  chromatin  skein  regular  and  uniform,  the  greatly  entangled  nature 
of  the  spirem  would  not  appear  during  the  development  of  the  spindle. 

We  have  seen  that  the  identity  of  the  individual  chromosomes  is 
lost  from  observation  in  the  daughter  spirem,  and  the  question  bear- 
ing upon  the  theory  of  the  individuality  of  the  chromosomes,  naturally 
arises  as  to  whether  the  chromosomes  of  the  second,  or  homotypic 
mitosis,  are  identical  with  the  pairs  of  granddaughter  segments  of  the 
anaphase  of  the  preceding,  or  heterotypic  division.  In  other  words, 
are  the  two  segments  of  each  chromosome,  appearing  in  the  nuclear 
plate  of  the  second  nuclear  division,  sisters  ?  Or  may  it  be  possible 
that  some  are  sisters,  while  others  are  composed  of  segments  from 
different  pairs  of  granddaughter  chromosomes  of  the  first  division  ? 


30 


INTRODUCTION. 


It  is  generally  conceded  that  the  segments  of  each  chromosome  are 
sisters,  and  it  is  conceivable  that,  no  matter  in  what  manner  or  when 
the  daughter  spirem  may  segment  during  division,  the  spindle  fibers, 
or  those  parts  of  the  cell  which  have  to  do  with  the  arrangement  of 
the  chromosomes  in  the  nuclear  plate,  are  able  to  bring  the  sister  seg- 
ments together  in  pairs. 

Strasburger,  Guignard,  and  others  regard  each  long  loop  or  turn  of 
the  daughter  spirem  as  representing  a  V  or  U  of  the  preceding  mitosis, 
and  that,  consequently,  the  spirem  segments  exactly  as  it  was  con- 
structed, i.  e.,  the  chromosomes  simply  separate  at  the  points  marking 
the  free  ends  of  the  V's  and  U's.  The  spirem  accordingly  breaks  up 
into  pieces  equal  to  the  length  of  two  segments  or  two  granddaughter 
chromosomes.  It  is  claimed  by  Strasburger  (1900,  pp.  23,  24)  that 
these  V's  or  U's  are  fastened  to  the  spindle  in  the  same  manner  as  in 
the  first  division,  namely,  at  the  angles  or  at  the  place  of  bending. 

Theoretically,  there  may  be  little  objection  to  this  view.  The  vast 
majority  of  facts,  however,  show  that  there  is  no  such  regularity  in 
the  shape  of  the  chromosomes,  or  in  their  manner  of  attachment  to  the 
spindle.  We  have  seen  that,  in  the  daughter  nucleus,  the  identity  of 
the  individual  chromosomes  cannot  be  recognized,  and  we  do  not 
know  whether  the  spirem  segments  in  the  same  manner  in  which  it 
was  constructed. 

But  if  the  spirem  should  segment  by  transverse  division  at  the  points 
marking  the  angles  of  the  V-shaped  chromosomes  instead  of  at  the  free 
ends,  then  it  is  clear  that  the  two  segments  of  each  chromosome  would 
not  be  sisters.  The  result  might  be  that  two  or  more  sister  chromo- 
somes would  go  to  the  same  daughter  nucleus,  a  condition  that  might 
furnish  a  basis  for  greater  vai'iation.  We  cannot  prove  either  propo- 
sition, and  the  author  is  not  disposed  to  enter  into  any  speculation  here 
upon  the  subject.  The  observed  facts  are  these :  The  identity  of  the 
individual  chromosomes  is  lost  in  the  daughter  nucleus,  and  we  do  not 
know  whether  the  segments  of  the  respective  chromosomes  appearing 
in  the  nuclear  plate  of  the  second  mitosis  are  sisters  or  not.  There  is 
also  no  basis  in  fact  for  the  conclusion  that  one  chromosome  is  heredi- 
tarily different  from  another. 

The  first  two  nuclear  divisions  in  the  embryo-sac  mother-cell,  so  far 
as  is  known,  are  quite  similar  and  homologous  to  those  in  the  pollen 
mother-cell.  In  Lilium  marlagon^  the  species  more  carefully  investi- 
gated by  the  author,  there  is  no  important  difference  in  the  behavior  of 
the  chromosomes.  It  may  be  mentioned,  moreover,  that  the  daughter 
nuclei  resulting  from  the  first  mitosis  approach  more  closely  the  resting 
condition  than  in  the  pollen  mother-cell. 


CELL-DIVISION.  3 1 

The  question  now  remains  whether  in  all  micro-  and  macro-spore 
mother-cells  of  the  higher  plants  a  double  longitudinal  splitting  of  the 
chromatin  takes  place  during  the  first  mitosis  and  how  prevalent  such 
a  phenomenon  is  in  both  plants  and  animals. 

In  those  plants  in  which  the  daughter  nucleus  passes  into  the  struc- 
ture of  the  complete  resting  stage,  it  is  certainly  difficult  to  understand 
the  significance  of  the  double  longitudinal  splitting  of  the  chromosomes 
in  the  first  division. 

CELL-DIVISION. 
THE  TYPE  OF  THE  HIGHER  PLANTS. 

Modern  research  has  established  the  very  important  fact  that  new 
cells  are  formed  from  uninucleate  or  multinucleate  mother-cells  accord- 
ing to  different  methods,  depending  largely  upon  the  manner  in  which 
the  new  plasma  membranes  differentiating  the  cells  are  formed. 

(I.)  Among  the  higher  plants,  and  some  Thallophyta  as  well,  in 
which  cell-division  is  generally  intimately  associated  with  nuclear 
division,  the  new  plasma  membrane  or  membranes  are  laid  down 
through  the  instrumentality  of  kinoplasmic  connecting  fibers,  extending 
between  the  nuclei  concerned. 

(2.)  In  the  ascus  of  certain  Ascomycetes^  where  the  new  cells 
(spores)  are  carved  out  of  a  common  nucleated  mass  of  cytoplasm  or 
mother-cell,  the  plasma  membrane  is  also  formed  by  kinoplasmic 
fibers,  but  these  are  polar  radiations  and  not  connecting  fibers.  The 
entire  plasma  membrane  of  such  cells  is  new,  that  of  the  mother-cell 
taking  no  part  in  the  process.  This  is  typical  and  real  free  cell- 
formation. 

(3.)  Another  form  of  cell-division  is  found  among  the  Myxomycetes 
and  certain  Phy corny cetes^  in  which  the  new  plasma  membranes  arise 
by  a  process  of  progressive  cleavage,  beginning  at  the  surface,  with  or 
without  any  connection  with,  or  aid  of,  vacuoles.  Kinoplasmic  con- 
necting fibers  or  radiations  are  in  no  way  connected  with  this  process. 
This  type  we  may  know  as  cell-cleavage.  It  resembles  the  cleavage  of 
animal  cells  more  closely  than  do  the  other  processes  of  cell-formation 
in  plants. 

(4.)  There  is  yet  another  method  of  cell-formation  typified  by 
Dictyota  and  Stypocaulon  among  the  brown  algae,  in  which  the  new 
plasma  membrane  seems  to  be  a  direct  transformation  of  the  meshes 
or  threadwork  of  the  cytoplasm.  It  is  not  a  cleavage  like  the  last 
mentioned,  nor  are  any  connecting  fibers  present  to  take  part  in  the 


32 


INTRODUCTION.  ' 


formation  of  the  cell-plate.  This  method  is,  however,  closely  related 
to  cleavage. 

As  an  illustration  of  the  method  of  cell-plate  formation  typical  of 
higher  plants,  the  pollen  mother-cells  of  Lilium  furnish  excellent 
material.  Here  a  cell-division  follows  the  first  nuclear  division.  The 
connecting  fibers  are  well  developed,  and  with  suitable  fixing  and 
staining  the  details  stand  otlt  with  a  clearness  unequaled  among  plants. 
As  we  have  seen  in  Fig.  lo,  G,  the  daughter  spirems  are  connected  by 
a  beautiful  system  of  connecting  fibers,  which  is  slightly  barrel-shaped 
at  an  early  stage.  The  fibers  soon  show  a  thickening  in  the  equatorial 
region,  which  stains  more  intensely  with  gentian  violet.  The  thicken- 
ings are  not  granular  or  lumpy,  but  rather  homogeneous,  and  are  due 
to  the  accumulation  of  kinoplasm,  the  substance  out  of  which  the 
cell-plate,  or  plasma  membrane,  is  made.  At  a  little  later  stage 
(Fig.  ID,  H)  there  appears  in  the  central  part  of  the  system  of  con- 
necting fibers  in  the  region  of  the  equator  a  fine  homogeneous  line, 
the  beginning  of  the  cell-plate.  This  young  cell-plate  is  evidently 
in  the  form  of  a  circular  disk,  which  proceeds  in  growth  uniformly 
toward  the  periphery  of  the  cell.  The  cell-plate  is  not  necessarily 
formed  by  the  meeting  or  union  of  thickened  places  of  the  connecting 
fibers,  for  in  many  cases  the  fibers  are  too  far  apart.  The  kinoplasmic 
material  is  brought  to  the  place  occupied  by  the  new  plasma  membrane 
and  there  deposited  in  the  form  of  a  fluid  substance.  With  the  further 
growth  of  the  cell-plate  the  connecting  fibers  bulge  out  more  and 
more,  being  always  thicker  and  more  numerous  at  the  outer  edge  or 
surface  of  the  system  (Fig.  lo,  H).  As  the  peripheral  fibers  of  the 
barrel-shaped  system  bulge  out,  its  longitudinal  axis  becomes  shorter, 
so  that  the  daughter  spirems  come  eventually  to  lie  in  the  center  of 
the  daughter  cells.  In  Fig.  lo,  I,  the  cell-plate  is  just  complete,  the 
peripheral  fibers  which  have  reached  the  plasma  membrane  of  the 
cell  being  more  numerous  there. 

The  cell-plate  or  plasma  membrane  is  now  seen  to  be  double,  and 
it  is  the  author's  opinion  that  the  new  plasma  membrane  is  formed 
double.  The  fact  that  each  daughter  or  granddaughter  cell,  when 
somewhat  shrunken  at  this  stage,  is  seen  to  possess  its  own  plasma 
membrane,  seems  to  support  this  view. 

Soon  after  the  formation  of  the  plasma  membranes,  a  cell-wall  is 
deposited  between  them.  Until  the  primordia  of  the  daughter  nuclei 
(Fig.  lo,  J)  are  provided  with  a  nuclear  membrane,  the  chromatin 
spirem  is  in  the  form  of  a  circular  disk  from  whose  margin  radiates  a 
zone  of  kinoplasmic  fibers  toward  the  equatorial  edge  of  the  cell.  In 
optical  section  this  zone  appears  as  a  bundle  of  fibers  on  the  right  and 


CELL-DIVISION.  33 

left,  whose  elements  diverge,  meeting  the  concave  plasma  membrane 
at  different  points.  Other  delicate  fibers  extend  from  the  spirem  in 
all  directions  toward  the  plasma  membrane.  As  soon  as  the  nuclear 
membrane  appears  these  radiating  fibers  become  more  uniformly  dis- 
tributed about  the  nucleus.  They  undoubtedly  take  part  in  the  forma- 
tion of  the  spindle  in  the  division  of  the  daughter  nucleus. 

FREE  CELL-FORMATION. 

The  most  beautiful  and  best  known  illustration  of  typical  free  cell- 
formation  is  found  in  the  development  of  the  spores  in  the  ascus  of 
certain  Ascomycetes  as  described  by  Harper. 

The  delimination  of  the  spores  from  the  cytoplasm  in  Erysiphe  fol- 
lows immediately  after  the  close  of  the  last  of  the  three  successive  nuclear 
divisions  which  furnish  the  eight  nuclei  for  the  spores.  The  entire 
process  is  accomplished  by  those  kinoplasmic  fibers  which  constitute 
the  polar  radiations  of  the  last  nuclear  division  and  in  a  manner  quite 
peculiar  to  asci. 

All  of  the  eight  nuclei  pass  through  the  anaphase  at  the  same  time, 
and,  when  in  the  resting  condition,  cannot  be  distinguished  one  from 
the  other,  with  the  exception  of  those  that  He  close  to  the  wall.  The 
polar  radiations  persist  in  connection  with  those  nuclei  that  form 
spores,  while  from  those  which  do  not  the  radiations  disappear  entirely. 
The  chromatin  lies  mostly  free  in  the  nuclear  cavity,  but  it  is  always 
in  communication  with  the  nuclear  membrane,  especially  near  the 
centrosphere  (Fig.  12,  A).  As  the  first  indication  of  cell-formation, 
the  nucleus  becomes  pointed  and  develops  a  beak-like  prolongation  on 
the  side  next  to  the  pole  or  centrosphere.  This  point  or  beak  gradually 
elongates,  so  that  the  centrosphere  becomes  farther  removed  from  the 
body  of  the  nucleus  (Fig.  12,  B).  As  soon  as  the  beak  reaches  a 
length  which  exceeds  slightly  the  diameter  of  the  nucleus,  its  growth 
ceases.  This  beak  consists  not  of  a  single  fiber  or  thread  but  of  a 
slender  cylindrical  tube  arising  abruptly  from  a  rather  broad  base. 
Into  the  tube  there  extends  quite  to  the  centrosphere  a  continuation  of 
the  chromatin  net,  by  which  the  latter  remains  in  communication  with 
the  centrosphere.  In  the  base  of  the  beak  the  nuclear  network  is 
loose  and  more  open,  while  in  the  slender  part  it  is  drawn  out  into  a 
single  and  twisted  thread. 

As  soon  as  the  beak  has  reached  its  definitive  length  the  kinoplasmic 
radiations  undergo  a  remarkable  change.  The  radiations  which  have 
a  direction  similar  to  that  of  the  beak  begin  now  to  bend  or  grow 
backward,  with  the  centrosome  as  a  center,  toward  the  nucleus,  so  that 


«4  INTRODUCTION. 

the  aster  is  converted  into  a  hollow  cone  whose  apex  is  the  centro- 
sphere.  Neighboring  radiations  unite  and  grow  rapidly  in  length,  at 
the  same  time  bending  back  toward  the  nucleus  in  a  manner  resem- 
bling the  spray  from  a  fountain.  An  optical  section  of  this  stage  is 
shown  in  Fig.  12,  C.  With  further  growth  the  kinoplasmic  rays  give 
rise  to  a  sort  of  bell-shaped  or  half-ellipsoidal  structure  whose  center 
is  occupied  by  the  nucleus  and  whose  pole  is  formed  by  the  centro- 
some  (Fig.  12,  D).  Near  the  centrosome  the  fibers  have  already  formed 
a  continuous  but  extremely  thin  layer,  the  plasma  membrane,  separat- 
ing the  cytoplasm  of  the  spore  from  that  of  the  ascus.     At  the  edge  of 


Fig.  12.— Free  cell-formation  in  ascus  oi  Erysiphe  communis. 

A,  nucleus  with  centrosphere. 

B,  development  of  nuclear  beak. 

C,  polar  radiations  extend  outward  and  backward  as  spray  from  a  fountain. 

D,  formation  of  plasma  membrane  from  end  of  beak  outward,  and  continued  growth  of  kinoplasmic 

fibers  backward. 

E,  F,  meeting  of  fibers  at  opposite  end  of  ellipsoidal  spore  and  establishment  of  a  complete  plasma 

membrane  delimiting  spore-plasma  from  remaining  plasma  of  ascus. — (After  Harper.) 

the  bell  the  radiations  end  as  free  fibers,  continuing  their  growth,  how- 
ever, in  a  direction  corresponding  to  the  periphery  of  the  ellipsoid 
(Fig.  12,  E).  Finally  these  fibers  meet  in  a  point  which  is  directly 
opposite  the  centrosome,  and  unite  end  to  end  and  laterally.  The  for- 
mation of  the  plasma  membrane  continues,  so  that  eventually  an  ellip- 
soidal or  oval  cell  is  delimited  from  the  cytoplasm  of  the  ascus  by 
a  complete  plasma  membrane  (Fig.  12,  F).  At  first  the  plasma 
membrane  is  thicker  near  the  centrosome,  but  later  its  thickness  be- 
comes uniform  throughout. 


CELL-DIVISION. 


35 


Fig.  13,  I,  J,  shows  several  stages  of  the  process  just  described  in 
two  asci  of  Lachnea  scutellata. 

While  this  is  taking  place  the  nuclear  beak  becomes  smaller  and 
smaller  until  it  is  finally  reduced  to  a  mere  thread  in  which  chromatin 
and  membrane  are  no  longer  recognizable.  The  centrosome  remains 
for  a  short  time  as  a  deeply  staining  and  sharply  defined  disk  adhering 
to  the  plasma  membrane.  Very  soon  it  becomes  free  from  the  mem- 
brane and  is  drawn  back  to  the  somewhat  pointed  nucleus,  wliere  it 
appears  as  a  saddle-like  thickening  upon  the  point  of  the  nucleus,  or 


Fig.  13. — Free  cell-formation  in  the  ascus. 
G,  H,  Erysipke  communis.     I,  J,  Lachnea  scutellata. 
G,  the  plasma  membrane  is  complete ;   nuclear  beak  withdrawn  and  centrosome  saddle-shaped,  and 

closely  applied  to  the  nuclear  membrane. 
H,  a  mature  spore  with  cell-wall ;  centrosome  closely  applied  to  nuclear  membrane  at  upper  side. 
I,  J,  portions  of  two  asci  showing  several  steps  in  process  of  free  cell- formation  in  situ. — (After  Harper.) 

as  a  simple  disk  (Fig.  13,  G,  H).  The  nucleus  now  gradually  assumes 
its  original  spherical  form,  the  chromatin  passing  into  the  structure  of 
the  resting  stage,  while  the  centrosome  remains  closely  adhering  to 
the  nuclear  membrane. 

It  will  be  observed  that  in  the  specific  case  of  cell-formation  described 
the  plasma  membrane  is  completed  before  the  nucleus  has  reached  the 
resting  stage,  but  in  Lachnea  (Harper,  1900)  the  daughter  nuclei  of 
the  eight-nucleated  stage  are  completely  reconstructed  before  the  beaks 
are  formed.  This  may  be,  of  course,  a  case  of  individual  variation 
and  of  only  secondary  importance. 


36 


INTRODUCTION. 


CELL-CLEAVAGE. 

The  process  of  cell-formation  by  means  of  a  progressive  cleavage  is 
best  known  at  present  in  certain  Phycomycetes  and  Myxomycetes.  As 
a  convenient  and  suitable  illustration  of  this  method  the  process  of 
cleavage  leading  to  spore  formation  in  the  sporangium  of  Synchitrium^ 
parasitic  upon  the  hog  peanut,  and  of  Sforodinia  is  selected.  For 
our  knowledge  of  cleavage  we  are  again  indebted  to  the  researches  of 
Harper  ('99). 

The  so-called  initial  cell  of  the  sporangium  of  Synchitrium^  when 
almost  fully  developed,  is  large  enough  to  be  visible  to  the  unaided 
eye,  and  contains  a  relatively  large  nucleus  (Fig.  14,  A).  This  nucleus 
divides  several  times  until  a  large  number  of  nuclei  are  present,  which 
lie  irregularly  distributed  in  the  cytoplasm. 

Cleavage  of  the  cytoplasm  now  begins.  It  does  not  take  place  by 
repeated  bipartitions,  nor  by  the  simultaneous  precipitation  of  a  cell- 
wall  about  each  nucleus.  As  mentioned  in  a  preceding  paragraph,  it 
resembles  in  a  large  measure  the  process  in  certain  animals,  as  for 
example,  the  dividing  protoplasm  of  the  germinal  disk  of  the  chick, 
or  perhaps  more  nearly  that  in  certain  insect  eggs  in  which  a  series  of 
nuclear  divisions  precedes  cytoplasmic  segmentation.^ 

The  cleavage  begins  by  the  formation  of  furrows  on  the  surface, 
which  grow  deeper  and  deeper  in  a  direction  more  or  less  radial.  It 
is  progressive  and  divides  the  cell  into  successively  smaller  portions 
(Fig.  14,  D).     The  process  is  described  in  detail  by  Harper  as  follows  : 

These  grooves  are  in  reality  so  narrow  as  to  appear  as  plates,  which  grow 
wider  by  additions  along  their  inner  margins  till  they  intersect,  and  thus  divide 
the  protoplasm  into  irregular  blocks  or  sometimes  pyramids  with  their  bases  in 
the  surface  of  the  initial  cell  (Fig.  14,  D,  E).  Only  at  the  very  periphery  the 
separation  of  the  cut  surfaces  of  the  protoplasm  to  form  a  shallow  notch,  as 
it  appears  in  section,  reveals  the  true  nature  of  the  process  as  a  pushing  in  of 
the  free  surface  to  form  a  deep  though  extremely  narrow  constriction. 

In  many  cases  there  is  at  first  no  separation  of  the  newly  formed  surfaces  ; 
they  remain  closely  appressed,  up  to  the  periphery  of  the  cell.  The  groove 
appears  in  section,  merely  as  a  single  line  which  the  Zeiss  appochromatic  lens 
1.40  ap.  fails  to  resolve  into  two  closely  appressed  surfaces  (Fig.  14,  B).  The 
position  of  the  line  is  further  emphasized  by  the  arrangement  of  the  vacuoles, 
which  are  pushed  aside  and  form  in  section  two  more  or  less  regular  rows  in 
the  plane  of  the  newly  formed  surfaces  on  each  side  of  the  furrow.  Such  a  line 
might  be  taken  for  a  cell-plate  which  subsequently  splits  to  form  the  boundaries 
of  the  protoplasmic  segments  or  which  is  metamorphosed  into  the  cellulose 
walls  of  the  spores.  That  this  line,  however,  in  reality  represents  from  the  start 
two  closely  appressed  surfaces  is  abundantly  shown  in  many  cases. 

>  Hertwig :  Die  Zelle  und  die  Gewebe,  p.  187. 


CELL-DIVISION. 


37 


These  lines  of  cleavage  are  not  meridional  furrows  which  divide  the 
cell  symmetrically,  but  they  intersect  each  other  at  varying  angles, 
marking  off  the  surfaee  of  the  cell  by  a  network  of  grooves,  in  which 
the  meshes  are  of  an  irregular  shape  and  of  unequal  dimensions  (Fig. 
H,  E). 


Fig.  14.— Cell-cleavage  in  Synchiirintn  discipens . 


A,  sporangium  mother-celt. 

B,  Portion  of  cell  showing  two  nuclei  and  two  surface  cleavage-furrows. 

C,  multinucleate  stage,  showing  progressive  cleavage  by  furrows  from  surfece. 

D,  median  section  showing  cleavage  further  advanced. 

E,  section  from  surface  of  cell  in  early  stage  of  cleavage. 

F,  cell  after  segmentation  is  completed,  showing  uninucleate  protospores.— (After  Harper.) 

The  cleavage  is  progressive  from  the  surface  inward,  the  furrows  deepening 
in  general  in  a  radial  direction.  Still  they  may  be  curved,  and  are  inclined 
to  each  other  at  very  varying  angles  and  frequently  form  intersections  at  points 
near  the  surface  of  the  cell,  thus  cutting  off  superficial  blocks  of  protoplasm  of 
varying  shapes  and  sizes  (Fig.  14,  C),  so  that  we  have  a  central  solid  mass  or 


38  INTRODUCTION. 

cell  of  protoplasm  surrounded  by  a  layer  of  superficial  cells  ;  in  other  cases  the 
furrows  grow  radially  inward  without  intersecting  till  near  the  centre,  thus  form- 
ing narrow  cones  and  pyramids  with  their  bases  outward  (Fig.  14,  D). 

With  the  progress  of  cleavage  the  contraction  of  the  protoplasm  in 
Synchitriutn  becomes  very  noticeable,  the  furrows  open  widely  and 
the  masses  tend  to  become  rounded.  The  cell  is  thus  split  up  into  a 
number  of  blocks  of  varying  size  and  containing  a  variable  number  of 
nuclei.  In  these  large  cells  or  portions  of  protoplasm  cleavage  fur- 
rows show  no  tendency  to  orient  themselves  with  reference  to  the 
nuclei,  but  as  the  process  advances  and  the  pieces  become  smaller  the 
nuclei  are  seen  to  be  more  evenly  distributed.  Finally,  the  result  is 
always  the  separation  of  the  cytoplasm  into  uninucleate  masses  or 
cells  (Fig.  14,  F). 

It  is  interesting  to  note  that  the  process  which,  in  the  beginning, 
seemed  to  be  independent  of  the  nuclei,  is  finally  directed  solely  from 
the  standpoint  of  their  distribution. 

From  this  process  of  cleavage  in  Synchitrium  it  is  at  once  appar- 
ent that  we  have  a  method  of  cell-formation  which  is  fundamentally 
different  from  either  of  the  two  methods  described  in  the  preceding 
pages.  Here  there  ai-e  no  kinoplasmic  fibers  developed  in  connection 
with  the  nuclei  under  whose  instrumentality  plasma  membranes  are 
formed,  and,  in  earlier  stages  of  cleavage  in  the  sporangium,  new 
plasma  membranes  seem  to  be  developed  independently  of  nuclei, 
though  not  in  their  absence. 

In  certain  cases  of  cell-formation  by  cleavage,  in  which  very  large 
multinucleate  masses  of  protoplasm  are  involved,  as  in  the  plasmodium 
of  certain  Myxomycetes  and  in  sporangia  of  such  Phycomycetes  as 
Piloholus  and  Sporodinia^  vacuoles  play  a  very  important  part  either 
directly  or  indirectly. 

The  first  indication  of  the  cleavage  which  is  preparatory  to  the  for- 
mation of  the  columella- wall  in  the  sporangium  of  Pilobolus  (Harper, 
'99)  is  seen  in  the  gradual  appearance  of  a  layer  of  vacuoles  larger 
than  the  rest,  and  lying  in  the  curved  surface  which  marks  the  outline 
of  the  columella : 

The  vacuoles  become  flattened  in  their  radial  axes  parallel  to  the  surface  of 
the  sporangium,  and  form  thus  disk-like  openings  which  tend  to  fuse  at  their 
edges.  At  the  same  time  a  circular  cleft  is  seen  to  start  from  the  edge  of  the 
sporangiophore  opening  .  .  .  and  to  develop  upward,  cutting  into  the 
vacuoles,  so  that  they  become  connected  into  a  continuous  furrow  (Fig.  15,  A). 
Whether  this  furrow  is  continued  upward  to  enclose  the  whole  dome-shaped 
columella,  or  whether  the  vacuoles  in  the  upper  portion  fuse  edge  to  edge  before 
the  cleft  reaches  them,  is  difficult  to  determine.  The  process  is  a  progressive 
one,  the  cleavage  being  complete  in  certain  portions  sooner  than  in  others,  and 


CKLL-DIVISION. 


39 


at  a  very  late  period  strands  of  protoplasm  are  seen  connecting  the  spore  plasma 
with  that  in  the  columella.  It  is  not  impossible  that  many  of  the  apparently 
disk-shaped  vacuoles  are  sections  of  curved  openings  which  burrow  through  the 
plasma  from  below  upwards.  Frequently  vacuoles  which  are  distinct  in  one 
plane  are  seen,  by  focussing  up  and  down,  to  lie  connected.  There  can  be 
little  doubt,  however,  that  a  considerable  part  of  cleavage  of  the  columella  is 
accomplished  by  flattening  and  lateral  fusion  of  originally  ellipsoidal  or  spheri- 
cal vacuoles  ;  that  is,  the  cleavage  is  not  entirely  by  a  furrow  from  the  plasma 


Fig.  15. — Cell-cleavage  in  sporangium  otPiloboltu  erystallinu*. — (After  Harper.) 

A,  median  section  at  stage  when  columella  is  forming. 

B,  section  of  spore-plasma  from  base  of  sporangium,  showing  surface  cleavage-furrows;  a,  sporangial 

wall. 

C,  section  of  portion  of  upper  part  of  a  sporangium,  showing  irregular  sausage-shaped  bodies  formed  by 

cleavage  of  spore-plasma. 
D,  similar  to  C,  but  older,  showing  uninucleate  masses  (protospores). 

membrane  at  the  mouth  of  the  sporangiophore,  but  is  at  least  in  part  a  process 
of  separation  by  excretion  of  a  liquid  into  vacuoles  and  their  fusion  side  by  side 
in  situ.  These  vacuoles  are  not  situated  on  the  extreme  boundary  of  the  pro- 
toplasm adjacent  to  the  large  central  vacuole,  but  placed  where  the  dense  spore- 
plasma  first  becomes  characteristically  spongy.  At  the  base  of  the  sporangium 
indeed,  they  cut  through  plasma  as  dense  as  the  densest  spore-plasma  of  the 
sporangium.  Why  the  cell-wall  of  the  columella  could  not  be  deposited  on  the 
surface  of  the  central  vacuole,  as  well  as  on  the  surface  of  the  small  vacuoles, 


40  INTRODUCTION. 

and  thus  enclose  all  the  protoplasm  in  the  sporangium,  is  an  interesting  ques- 
tion. The  necessity  is  evident  that  the  cleavage  should  proceed  through  a 
tolerably  dense  plasma,  and  this  is,  perhaps,  due  to  the  need  of  two  proto- 
plasmic surfaces  in  contact  in  order  to  form  a  cell-wall. 

The  fact  that  the  columella  is  not  deposited  on  the  surface  of  the 
central  vacuole  seems  to  indicate  that  the  limiting  layer  of  a  vacuole 
is  not  quite  a  plasma  membrane,  although  it  may  partake  partly  of 
the  real  nature  of  one.  Although  there  is  much  to  show  that  the  wall 
of  a  vacuole,  such  as  we  are  dealing  with  here,  and  a  plasma  mem- 
brane are  closely  related,  yet  the  author  is  not  quite  ready  to  admit 
that  they  are  the  same.  Why  two  plasma  membranes  should  be  in 
contact  in  order  to  form  a  cell- wall,  as  suggested  by  Harper,  is  not 
quite  clear  to  the  author,  since  in  many  cases  a  single  plasma  mem- 
brane will  secrete  a  cell-wall. 

In  the  cleavage  of  the  spore-plasma,  which  begins  soon  after  the 
columella  is  complete,  vacuoles  also  take  an  important  part.  The 
cytoplasm  becomes  somewhat  vacuolar,  and  the  numerous  nuclei  are 
rather  evenly  distributed  throughout  its  mass.  Cleavage  furrows 
appear  now  near  the  base  of  the  sporangium,  cutting  the  surface  into 
irregular  polygonal  areas  (Fig.  15,  B).  At  the  same  time  vacuoles 
in  the  interior  become  angular,  appearing  three-cornered  in  section, 
and  their  edges  cut  through  the  cytoplasm  to  meet  similar  cleavage 
furrows  from  adjacent  vacuoles  (Fig.  15,  B).  In  the  meantime  the 
surface  furrows  which  have  been  growing  deeper  meet  and  become 
continuous  with  the  edges  of  the  vacuoles.  By  pressure  of  the  adja- 
cent plasma-masses,  the  surfaces  of  the  vacuoles  which  were  formerly 
convex  become  concave,  and  the  vacuoles  appear  as  intercellular 
spaces  between  the  cleavage-segments.  In  this  manner  the  spore- 
plasma  is  marked  out  into  irregular  blocks,  apparently  without  refer- 
ence to  the  size  or  number  of  nuclei  they  contain.  A  continuation  ot 
the  process  cuts  the  spore-plasma  into  oblong  rounded  sausage-shaped 
masses  containing  generally  two  to  four  nuclei  in  a  row  (Fig.  15,  C). 
These  oblong  masses  now  divide  transversely  to  form  rounded  bodies 
with  one  or  few  nuclei  (Fig.  15,  D).  This  completes  the  primary 
cleavage  by  which  the  spore-plasma  has  been  cut  up  into  smaller 
units  with  one  or  few  nuclei.  These  units  are  not  the  spores.  They 
undergo  a  period  of  growth  and  nuclear  division  before  the  final 
cleavage  divisions  take  place  by  which  the  mature  spores  are  pro- 
duced. The  last  divisions  are,  however,  similar  to  the  first,  presenting 
the  simpler  process  of  cleavage  or  fission. 

In  the  sporangium  of  Pilobolus,  we  have  a  cleavage  which  is  of 
the  same  type  as  in  Synchitrium.,  with  the  exception  of  the  promi- 


CKLL-DIVISION.  4I 

nent  part  taken  by  the  vacuoles  in  the  former.  Although  the  mem- 
branes of  these  vacuoles  may  not,  at  first,  be  exactly  similar  to  plasma 
membranes,  they  are  undoubtedly  converted  into  them.  Since  we 
assume  that  the  plasma  membrane  is  largely  of  a  kinoplasmic  nature, 
and  attribute  to  it  something  of  a  morphological  rank  in  the  cell,  it 
may  not  be  wholly  fanciful  to  suggest  that  the  limiting  membrane  of 
a  vacuole  may  be  developed  into  a  real  plasma  membrane,  and  that 
this  actually  takes  place  in  the  plants  in  question. 

CELL-DIVISION  IN  DICTYOTA  AND  STYPOCAULON. 

There  is  yet  another  method  of  cell-formation  which  has  been 
observed  in  certain  of  the  brown  algae  that  differs  materially  from  the 
process  of  cleavage  already  described.  There  are  no  kinoplasmic 
connecting  fibers  by  which  a  plasma  membrane  may  be  formed,  nor 
is  it  a  cleavage  such  as  has  been  described  for  certain  fungi. 

The  plasma  membrane,  or  cell-plate,  seems  to  be  formed  directly 
out  of  the  apparently  undifferentiated  framework  of  the  cytoplasm. 
This  type  of  cell-formation  has  been  observed  in  such  Phceophycece 
as  Stypocaulon  (Swingle,  '97),  Fucus  (Strasburger,  '97),  and  Dic- 
tyota  (Mottier,  1900). 

Swingle  has  followed  the  development  of  the  cell-plate  in  great 
detail  in  the  apical  cell  of  Stypocaulon.  Here  each  division  of  the 
nucleus  is  followed  by  a  cell-division.  The  bulk  of  the  cytoplasm 
presents  a  very  beautiful  and  typical  alveolar  structure,  and  the  first 
indication  of  a  cell-plate  is  seen  in  certain  alveolae,  which  show  a 
tendency  to  arrange  themselves  across  the  cell  in  a  transverse  plane 
(Fig.  16,  B).  As  soon  as  this  orientation  of  the  alveolae  becomes  more 
marked,  the  transverse  alveolar  lamellae  form  a  more  continuous  plane 
which,  in  section,  appears  as  a  very  fine  line.  During  these  changes 
neither  an  increase  in  the  number  of  connecting  fibers  between  the 
nuclei  nor  any  perceptible  change  whatever  in  the  arrangement  of  the 
kinoplasm  was  to  be  seen.  Only  a  few  fibers  or  lines  of  force,  indi- 
cated by  the  arrangement  of  the  alveoloe  of  the  frothy  plasma,  extend 
from  the  nucleus  of  the  apical  cell  to  the  seat  of  cell-plate  formation, 
and  fewer  still  from  the  lower  nucleus  to  the  same  place.  It  is  certain 
that  if  there  be  real  fibers,  they  must  be  extremely  delicate  and  not 
numerous  enough  to  lead  one  to  suppose  that  the  cell-plate  is  laid  down 
by  any  such  process  as  in  the  higher  plants. 

The  author  has  found  that  the  development  of  the  plasma  membrane 
in  the  tetraspore  mother-cell  of  Dictyota  (Mottier,  1900)  is  similar  to 
that  of   Stypocaulon.     Here  there  is  absolutely  no  visible  trace  of 


42 


INTRODUCTION. 


kinoplasmic  connecting  fibers  between  the  nuclei,  and  in  the  region  of 
the  cell-plate  the  cytoplasm  seems  undifferentiated.  The  plasma  mem- 
branes, or  cell-plates,  which  will  separate  the  four  spores,  are  laid 
down  almost  simultaneously.  In  the  region  where  they  are  to  appear 
the  cytoplasm,  as  elsewhere,  except  near  the  nuclei,  presents  the  same 
visible  structure  of  alveolae,  or  perhaps  a  mixture  of  alveolae  and  a 
thread-like  network.     Rather  large  and  small  meshes  are  intermingled. 


Fig.  i6. — Cell-plate  oi  Dictyota  dichototna  and  Stypocaulon. 

A,  portion  of  cell-plate  from  tetraspore  mother-cell  of  Dictyota,  formed  apparently  by  arrangement  of 
alveolar  lamellae  into  a  continuous  and  even  plane. 

B,  same  from  apical  cell  of  Stypocaulon, — (B,  after  Swingle.) 

The  small-meshed  structure  is  apparently  more  granular  than  that 
with  larger  meshes. 

The  first  visible  trace  of  a  cell-plate  is  manifested  by  the  transverse 
walls  of  the  alveolae  becoming  perceptibly  thicker  and  arranging  them- 
selves in  such  a  way  as  to  appear  as  an  uneven  or  somewhat  zigzag 
line  in  section  (Fig.  i6,  A).  In  this  cell-plate  primordium  the  walls 
of  both  large  and  small  meshes  take  part.  At  first  certain  of  the  alve- 
olar  lamellae  are  thinner  than   others,  so  that   the    cell-plate    seems 


CELL-DIVISION. 


43 


interrupted  at  these  places,  but  eventually  and  gradually  it  attains  a 
uniform  thickness.  Very  soon  the  cell-plate  is  a  uniform  plane, 
appearing  in  section  as  a  rather  smooth  line. 

The  cell-plate  is  not  always  laid  down  everywhere  simultaneously, 
but  sometimes  it  appears  at  first  more  marked  at  the  periphery.  This 
seems  to  depend  upon  the  position  of  the  nuclei.  It  is  evident  that  in 
Dictyota  no  differentiated  kinoplasmic  connecting  fibers  can  be  recog- 
nized by  which  the  cell-plates  are  formed.  It  seems  that  the  appar- 
ently undifferentiated  framework  of  the  cytoplasm,  consisting  of  large 
and  small  meshes  in  the  immediate  region  of  the  cell-plate,  is  con- 
verted into  a  plasma  membrane.  The  cell-plates  are  certainly  formed 
under  the  influence  of  the  nuclei,  and  kinoplasm  in  some  form  enters 
into  the  process. 

The  behavior  of  the  cell-plate  toward  certain  stains,  particularly 
gentian  violet,  and  the  character  and  behavior  of  the  cytoplasm  in  that 
region,  immediately  preceding  the  appearance  of  the  plasma  membrane, 
strongly  suggests  that  the  latter  is  not  an  actual  transformation  of  the 
alveolar  walls,  but  that  the  substance  of  the  cell-plate  is  deposited  by 
kinoplasm  present  in  the  framework  of  the  cytoplasm.  The  form  in 
which  this  kinoplasm  occurs  here  is  difficult  to  determine,  but  it  mat- 
ters very  little  whether  it  takes  on  the  form  of  a  fibrous  network  or 
of  alveolae,  or  whether  it  is  present  merely  as  a  homogeneous  fluid. 

Of  the  several  types  of  cell  formation  briefly  described  in  the  fore- 
going pages,  the  first,  or  that  which  is  typical  for  higher  plants, 
occurs  generally  in  all  plants  from  the  liverworts  up.  It  obtains  also 
in  Chara  and  Nitella  and  has  been  found  by  Fairchild  ('97)  in  Basi- 
diobolus.     This  method  doubtless  occurs  in  other  algae  and  fungi. 

The  process  of  typical  free  cell-formation,  as  found  in  the  ascus  of 
the  Ascomycetes  mentioned,  is,  so  far  as  known,  restricted  to  this 
group  of  fungi 

A  process  of  free  cell-formation  has  been  described  by  Strasburger 
in  the  egg-cell  of  Ephedra^  but  there  it  differs  considerably  from  that 
in  the  ascus,  since  centrosomes  or  centrospheres  are  not  present  and 
the  kinoplasmic  fibers  radiate  in  all  directions  from  each  nucleus. 

The  process  of  cleavage  is  the  method  of  cell-formation  in  the  plas- 
modium  of  Myxomycetes  and  in  certain  Phy  corny  cat  es.  It  is  also  of 
undoubted  occurrence  in  many  algae  and  in  other  fungi. 

Whether  the  kind  of  cell-plate  formation  described  for  Stypocaulon 
and  Dictyota  occurs  outside  of  the  brown  algae,  future  research  must 
determine. 

The  process  of  constriction  characteristic  of  Cladophora  and  Spiro- 
gyra  may  be  looked  upon  as  a  kind  of  cleavage  in  which  the  formation 


44  INTRODUCTION. 

of  the  new  cell-wall  is  gradual  and  progressive  from  the  old  cell-wall 
inward,  instead  of  being  developed  simultaneously  from  a  plasma 
membrane  previously  formed.  Whether  in  such  cases  new  plasma 
membranes  are  formed  across  the  ends  of  the  daughter  cells  which  come 
in  contact  with  the  new  transverse  cell-wall  the  author  is  unable  to 
state. 

THE  CENTROSOME  AND  THE  BLEPHAROPLAST. 

As  illustrations  of  karyokinesis  in  which  the  spindle  arises  through  the 
agency  of  centrospheres  I  have  selected  thetetraspore  mother-cell  of  Dic- 
tyota  and  the  ascus  of  certain  Ascomycetes^  because  the  centrosphere 
is  probably  best  known  in  those  cells  and  because  the  entire  develop- 
ment of  the  mitotic  figure  has  been  followed  in  great  detail.  In  these 
plants,  as  well  as  in  Fucus  and  certain  Sphacelariacece ,  we  have  seen 
that  the  body  which  we  call  a  centrosome  is  one  that  persists  from  one 
cell-generation,  or  nuclear  generation,  to  another  in  vegetative  and  in 
certain  reproductive  cells.  It  seems  to  be  capable  of  division,  and  is 
the  centre  of  radiations  that  give  rise  to  the  karyokinetic  spindle.  We 
do  not  know  with  absolute  certainty  that  the  centrosome  divides, 
although  the  evidence  seems  to  admit  of  no  other  interpretation. 

In  addition  to  the  plants  just  mentioned,  centrospheres  have  been 
found  in  some  liverworts,  in  diatoms,  and  in  certain  Rhodophycece. 
In  the  diatoms,  however,  the  behavior  of  the  centrosome  during  karyo- 
kinesis, as  described  by  Lauterborn  ('96),  differs  widely  from  the 
typical  cases  described  in  the  preceding  pages.  In  species  of  .Pinnu- 
laria,  Surirella^  and  others,  Lauterborn  finds  that  the  peculiar  cen- 
tral spindle  arises  from  the  centrosome  by  a  division  or  process  of 
budding.  "Es  scheint  mir  keinem  Zweifel  zu  unterliegen,  dass  die 
Anlage  der  Centralspindel  aus  dem  Centrosom  durch  eine  Theilung 
(oder,  wenn  man  lieber  will,  Knospung)  hervorgeht  "  (1.  c,  p.  61). 

In  the  diatoms  in  question  the  original  centrosome  is  a  relatively 
large  globular  body  which  is  the  center  of  a  system  of  beautiful  radia- 
tions. Soon  after  the  budding  off  of  the  primordium  of  the  central 
spindle,  the  original  centrosome,  with  its  radiations,  disappears,  and 
what  is  taken  to  be  the  new  centrosomes  arise  near  the  poles  of  the 
spindle  and  apparently  from  it. 

So  far  as  the  author  is  aware,  such  a  phenomenon  has  no  parallel 
among  plants,  and  it  is  impossible  to  bring  the  process  of  spindle- 
formation  in  the  diatoms,  as  described  by  Lauterborn,  into  line  with 
anything  known  in  other  organisms. 

When  we  consider  the  facts  alone  in  the  algae  and  fungi  mentioned, 
we  certainly  have  strong  evidence  in  favor  of  the  doctrine  of  the  genetic 


THE  CENTROSOME  AND  THE  BLEPHAROPLAST.         45 

continuity  of  the  centrosomes ;  but  from  the  fact  that  no  such  organs 
exist  in  the  higher  plants,  and  that  they  seem  to  be  wanting  in  many 
Thallophyta  as  well,  this  view  is  greatly  weakened,  if  not  rendered 
quite  untenable. 

On  the  zoological  side  of  the  question,  the  recent  researches  of  Wil- 
son (1901)  on  eggs  of  Toxopcnustes^  which  were  made  to  develop 
parthenogenetically  through  certain  stages  by  means  of  chemical 
stimuli,  throw  new  light  upon  the  subject.  In  segmenting  eggs 
induced  to  develop  parthenogenetically  by  means  of  a  treatment  with 
suitable  solutions  of  magnesium  chloride,  numerous  asters  (cytasters) 
often  made  their  appearance  in  the  cytoplasm  in  addition  to  the  nuclear 
asters.  Similar  asters  may  arise  also  in  non-nucleated  fragments  of 
eggs.  These  "  cytasters,"  just  as  the  segmentation  or  nuclear  asters, 
may  consist  of  a  very  distinct  centrosome  upon  which  is  centered  a 
system  of  beautiful  radiations.  The  centrosomes  divide,  and  a  central 
spindle  is  formed  between  the  daughter  centrosomes.  In  fact,  the 
"  cytasters  "  are  exactly  like  the  normal  cleavage-asters  arising  in  con- 
nection with  the  chromatin.  As  the  evidence  seems  conclusive  that 
the  "cytasters"  arise  de  novo,  Wilson  concludes  that  centrosomes 
occurring  normally  in  cells  arise  also  cie  novo,  and  that  the  doctrine 
of  the  genetic  continuity  of  the  centrosome  is  untenable. 

It  is  not  known  whether  anything  comparable  to  these  "  cytasters" 
ever  occurs  in  a  plant  egg-cell,  which  may  be  made  to  develop  parthe- 
nogenetically by  artificial  means,  and  consequently  we  cannot  accept 
the  conclusion  upon  this  basis  as  applicable  to  plants.  There  are, 
however,  in  plants  many  well  established  facts  which  argue  strongly 
against  the  view  that  the  centrosome  or  centrosphere  is  an  organ  of 
morphological  rank. 

In  1897,  the  author  made  the  unqualified  statement,  to  which  he 
still  adheres,  that  centrosomes  or  centrospheres  do  not  occur  in  the 
higher  plants,  and  nearly  all  research  since  made  along  this  line  has 
only  confirmed  this  view.  We  know  now  that  the  structures  which 
Guignard  so  beautifully  figured  in  1891  for  cells  of  Lilium  were  the 
product  of  preconceived  ideas  and  the  misinterpretation  of  certain 
facts.  There  are  still  a  few  observers  who  persist  in  seeing  centro- 
spheres  in  the  cells  of  higher  plants,  in  which  a  score  or  more  of  the 
most  competent  cytologists,  with  the  aid  of  the  very  best  methods, 
have  failed  to  find  any  such  structures.  It  may  be  of  some  interest  to 
note,  however,  that  the  centrospheres  figured  more  recently  by  these 
obsei-vers  are  not  drawn  with  the  old-time  diagrammatic  distinctness, 
and  it  will  probably  not  be  long  till  these  structures  will  not  appear 
at  all  in  figures  illustrating  karyokinetic  phenomena  in  Allium  cefa 
and  species  of  Lilium. 


46  INTRODUCTION. 

At  the  present  writing  it  is  the  opinion  of  the  author  that  individu- 
alized centrosomes  or  centrospheres  do  not  occur  in  plants  above  the 
liverw^orts,  and  they  are  certainly  absent  in  certain  species  of  these 
[Anthoceros).  On  the  whole,  these  structures  are  well  established  in 
only  a  few  Thallophyta. 

As  the  writer  has  already  stated  in  a  former  paper  (Mottier,  1900), 
if  we  take  into  consideration  only  "such  plants  as  Fucus^  Stypocaulon^ 
Dictyota^  and  certain  Ascojnycetes^  there  are  good  grounds  for  the 
view  that  the  centrosome  is  an  organ  of  morphological  value ;  but  the 
evidence  furnished  by  these  forms,  however  convincing  it  may  seem, 
is  not  quite  sufficient,  especially  in  the  light  of  our  knowledge  of  kary- 
okinesis  in  forms  in  which  centrosomes  or  centrospheres  have  not  been 
found ;  for  there  is  no  reason  for  believing  that  the  spindle  fibers  in 
plants  devoid  of  centrosomes  are  of  a  different  substance  from  the 
radiations  or  spindle  fibers  developed  in  connection  with  an  aster. 

Space  will  not  permit  of  a  discussion  of  such  questions  as  whether 
the  radiations  are  outgrowths  of  the  centrosome  considered  as  a  mor- 
phological unit,  or  constructed  out  of  the  kinoplasm  by  the  centrosome, 
or  whether  the  centrosome  is  only  a  denser  mass  of  kinoplasm,  formed 
by  the  meeting  of  the  polar  radiations,  and  which  may  persist  after  the 
radiations  and  spindle  fibers  have  disappeared.  It  may  be  stated  in 
this  connection  that  in  plants  there  is  little  to  support  the  view  that  the 
radiations  are  centripetal  or  centrifugal  currents.  They  do  not  seem 
to  be  currents  at  all.  We  understand  radiations  and  spindle  fibers  to 
be  fine,  more  or  less  homogeneous,  kinoplasmic  threads  which  are 
capable  of  contracting,  extending,  or  becoming  changed  into  a  uniform 
and  homogeneous  mass. 

We  have  now  to  consider  the  relation  of  the  centrosome  to  the 
blepharoplast^  or  cilia-bearer,  which  is  so  well  known  in  the  sperma- 
tozoid  of  the  Archegoniates  (see  Chapter  V). 

Belajeff,  Ikeno,  and  Hirase  and  a  few  others  regard  the  blepharo- 
plast  of  the  fern  and  certain  gymnosperms  as  the  homolog  of  the  centro- 
some. It  seems  to  the  author  that  such  a  conclusion  is  merely  a  hasty 
judgment,  which  does  violence  to  the  facts  as  they  are  known  at  present. 
The  development  and  function  of  the  blepharoplast,  as  will  be  seen 
from  the  chapter  referred  to,  shows  clearly  that  this  structure  lacks  the 
more  essential  distinguishing  characteristics  of  the  normal  centrosphere, 
as  it  is  known  in  the  cases  most  thoroughly  investigated.  The  bleph- 
aroplast is  not  the  center  of-  kinoplasmic  radiations  which  form  a 
karyokinetic  spindle.  So  far  as  has  been  shown  the  radiations  of  the 
blepharoplast  primordia  take  no  part  in  the  formation  of  the  spindle. 
These  primordia  do  not  divide  to  give  rise  to  new  blepharoplasts,  but 


THE  CBNTROSOME  AND  THE  BLEPH  AROPLAST.         4^ 

arise  de  novo.  They  do  not  persist  through  several  successive  genera- 
tions of  cells,  two  cell-generations  representing  the  maximum  time  of 
their  duration.  In  short,  the  blepharoplast  develops  merely  the  cilia 
and  forms,  therefore,  the  locomotary  apparatus  of  the  spermatozoid. 

No  phylogenetic  relationship  has  as  yet  been  shown  to  exist  between 
blepharoplast^' and  centrosome.  The  fact  is  that,  in  those  plants  in 
which  blepharoplasts  occur,  there  are  no  centrosomes  with  which  to 
show  any  phylogenetic  relationship.  The  main  reason,  it  seems,  for 
regarding  the  blepharoplast  as  the  homolog  of  the  centrosome  is  the 
sole  fact  that  the  primordia  of  the  former  at  a  certain  period  of  develop- 
ment are  provided  with  a  system  of  radiations,  giving  them  the  appear- 
ance of  centrospheres. 

The  view  concerning  the  origin  and  phylogeny  of  the  blepharoplast 
as  advanced  by  Strasburger  is  of  interest,  since  it  is  the  only  one  that 
seems  to  take  into  consideration  all  the  facts.  Strasburger  derives  the 
blepharoplast  from  the  cilia-bearer  of  the  zoospores  and  gametes  in  the 
algae.  In  the  zoospoi'es  of  certain  algae,  e.  g".^  Vaucherta^  (Edogo- 
nium^  and  others,  the  cilia  spring  from  a  localized  thickening  of  the 
plasma  membrane  (Hautschicht)  at  the  anterior  end.  In  (Edogonium 
this  kinoplasmic  thickening  is  in  the  shape  of  a  double  convex  lens, 
from  the  edges  of  which  arise  the  numerous  cilia.  In  the  large  swarm 
spore  of  Vaucheria  the  nuclei  seem  to  be  intimately  connected  with  the 
formation  of  the  cilia-bearer.  The  nuclei  migrate  to  the  plasma  mem- 
brane and  elongate  in  a  direction  at  right  angles  to  the  surface  of  the 
spore.  The  anterior  end  of  each  pear-shaped  nucleus  comes  in  contact 
with  the  plasma  membrane.  That  part  of  the  plasma  membrane  in 
contact  with  the  nucleus  thickens  in  the  form  of  a  delicate  concavo- 
convex  lens,  from  two  points  of  which,  on  opposite  sides,  spring  the  cilia. 
The  size  and  shape  of  the  cilia-bearer  vary,  of  course,  in  different 
algae.  Timberlake  ('oi)  finds  a  small  body  at  the  base  of  the  cilia  in 
Hydrodictyon^  but  it  does  not  seem  to  be  part  of  the  plasma  membrane. 

As  Strasburger  has  pointed  out,  the  "  mouth-piece  "  of  swarm  spores 
and  gametes  is  not  to  be  confounded  with  the  cilia-bearer,  since  the 
former  represents  the  entire  anterior  end  of  the  cell  free  from  chloro- 
phyll. It  is  true  that  the  cilia-bearer  is  not  well  known  in  the  sperma- 
tozoids  of  algae,  but  transitions  show  that  in  all  probability  the  sperma- 
tozoids  were  derived  from  male  gametes  which  in  every  way  resembled 
asexual  swarm  spores.  The  spermatozoids  of  Volvox  globator  are 
regarded  as  a  good  illustration  of  this  relation,  for  in  structure  they 
occupy  an  intermediate  position  between  the  gametes  of  algae  and  the 
spermatozoids  of  Chara.  In  Volvox  the  two  laterally  inserted  cilia 
would  seem  to  indicate  that  the  blepharoplast  had  undergone  a  lateral 


4$  INTRODUCTIOX. 

displacement,  for  the  entire  anterior  end  of  the  spermatozoid  of  Volvox 
is  certainly  not  blepharoplast.  (The  very  suggestive  theory  of  Stras- 
burger  carries  with  it  a  certain  degree  of  probability,  yet  to  what  extent 
it  is  true  further  research  must  determine.) 

If,  however,  any  genetic  relationship  exists  between  centrosome  and 
blepharoplast,  the  evidence  is  certainly  to  be  sought  in  the  lower 
plants.  In  this  connection  it  is  of  the  greatest  importance  to  know 
first  of  all  whether,  in  such  algae  as  the  Spkacelariacece^  in  which 
centrosomes  are  known,  any  relation  exists  between  the  centrosome 
and  cilia-bearer,  assuming,  of  course,  that  the  cilia  arise  here  also  from 
a  differentiated  body.  In  Chara  and  in  those  Archegoniates^  with 
blepharoplasts  no  centrosomes  are  found,  neither  is  any  such  body 
known  to  take  part  in  the  formation  of  the  spindle  in  such  algae  as 
(Edogonium^  and  others  in  which  highly  developed  cilia-bearers 
occur.  Although  these  facts  do  not  prove  anything,  yet  they  lend 
encouragement  to  the  belief  that  centrosome  and  blepharoplast  may  be 
homologous  structures,  or  in  some  degree  phylogenetically  related. 

Those  who  maintain  that  the  cilia-bearers  are  centrosomes  have  not, 
it  seems,  approached  the  question  from  the  standpoint  just  mentioned, 
but  seem  to  have  based  their  conclusion  upon  the  resemblance  between 
blepharoplast  primordia  and  centrospheres,  or  upon  analogies  between 
the  spermatozoids  in  plants  and  the  spermatozoa  of  certain  animals. 

Belajeff  ('99),  who  claims  that  blepharoplasts  are  homologous  with 
centrosomes,  strengthens  his  view  by  his  observations  in  spermagenous 
cells  of  Marsilia.  In  the  grandmother-cells  of  the  spermatozoids  of 
this  plant  he  finds  that  the  blepharoplast  primordia,  which  lie  some 
distance  from  the  nucleus,  divide  previous  to  the  division  of  the  nucleus, 
and  between  the  two  separating  daughter  primordia  a  small  central 
spindle  is  developed  just  as  in  certain  animal  cells.  From  this  small 
amphiaster  the  karyokinetic  figure  is  developed.  This,  if  true,  is  the 
first  case  on  record  in  plants  in  which  a  central  spindle  is  formed 
between  the  daughter  centrosomes,  lying  in  the  cytoplasm  some  dis- 
tance removed  from  the  nucleus. 

In  the  light  of  what  is  now  known  concerning  the  development  of 
the  spindle  in  Chara  and  in  the  Pieridophyta,  the  author  entertains 
serious  doubts  concerning  the  accuracy  of  Belajeff's  statement.  Oster- 
hout's  ('97)  studies  on  the  development  of  the  spindle  in  the  spore 
mother-cells  of  Equisetum  prove  beyond  all  question  that  centrosomes 
are  not  present  in  that  genus.  In  other  Pteridophyta  the  majority  of 
all  investigations,  which  have  been  thorough  or  reasonably  exhaustive, 
shows  that  centrosomes  or  centrospheres  are  absent  there  also. 

'  Marchtintia  folym«rfka  excepted. 


SIGNIFICANCE    OF    THE    SEXUAL   PROCESS.  49 

From  our  present  state  of  knowledge  of  the  development  of  the 
blepharoplast  there  is  but  one  conclusion,  it  seems  to  the  author,  that 
can  be  legitimately  drawn  concerning  their  origin,  namely,  that  they 
arise  de  novo.  As  regards  centrosomes  the  evidence  is  more  compli- 
cated and  conflicting.  Although,  in  the  opinion  of  the  author,  the 
evidence  is  decidedly  against  the  doctrine  of  the  genetic  continuity  of 
the  centrosome,  yet  the  proof  is  not  quite  conclusive.  If  centrosomes 
also  arise  de  novo,  then  the  problem  assumes  a  slightly  different  aspect, 
for  it  is  questionable  whether  we  are  justified  in  speaking  of  homologies 
between  organs  that,  as  such,  are  without  genetic  continuity. 

There  is  strong  evidence,  which  seems  to  be  increasing  from  day 
to  day,  that  it  is  the  fundamental  substance  known  in  the  plant  cell  as 
kinoplasm  which  is  genetically  continuous.  After  a  careful  considera- 
tion of  the  facts,  the  author  is  led  to  the  same  conclusion  concerning 
the  centrosome  to  which  he  gave  expression  in  1900,  in  a  paper  on 
the  nuclear  division  in  Dictyota  (1.  c,  p.  178),  namely,  that  it  is  the 
kinoplasm  which  should  hold  the  rank  of  morphological  unit,  and  that 
the  centrosome  should  be  regai'ded  as  an  individualized  part  of  the 
same,  existing  in  that  form  in  some  organisms  and  not  in  others,  for 
reasons  that  cannot  at  present  be  explained.  As  regards  blepharo- 
plasts,  about  the  only  conclusion  in  harmony  with  all  the  facts  is  that 
these  bodies  represent  individualized  parts  of  the  kinoplasm  which 
arise  de  novo  in  certain  spermagenous  cells,  and  from  which  the  cilia 
are  developed. 

SIGNIFICANCE   OF    THE  SEXUAL  PROCESS   AND    THE  NUMERI- 
CAL REDUCTION  OF  THE  CHROMOSOMES. 

Speaking  generally,  the  phenomena  resulting  from  the  sexual  process 
fall  into  two  categories,  namely,  (i)  the  transmission  of  hereditary 
characters,  together  with  the  blending  of  two  lines  of  descent  by  the 
fusion  of  the  sexual  nuclei,  and  (2)  the  imparting  of  a  growth  stimulus 
to  the  fecundated  egg  or  to  the  zygote,  by  which  the  energy  of  growth 
and  division  is  restored. 

Correlative  with  the  first  category  is  the  reduction  in  the  number  of 
chromosomes.  The  doctrine  of  the  significance  of  the  numerical 
reduction  of  the  chromosomes  now  generally  accepted  by  botanists  as 
a  working  hypothesis,  was  first  stated  in  a  well  organized  form  and 
presented  formally  to  botanical  science  by  Strasburger  ('94)  in  a  mas- 
terly essay  on  the  "  Periodic  Reduction  of  Chromosomes  in  Living 
Organisms."  The  enunciation  of  this  doctrine  marked  the  beginning 
of  a  new  epoch  in  the  study  of  sexuality  and  in  cytological  research  in 
plants. 


50 


INTRODUCTION. 


The  simplest  and  most  primitive  organisms  known  reproduce  them- 
selves asexually,  and  vv^e  are  obliged  to  assume  that,  from  a  phylo- 
genetic  standpoint,  sexually  differentiated  organisms  were  descended 
from  asexual  forms.  The  process  of  this  descent  is  clearly  illustrated 
by  certain  of  the  green  algse  in  which  the  sexual  act  consists  in  the 
fusion  of  exactly  similar  motile  gametes.  These  gametes  were  un- 
doubtedly derived  from  asexual  swarm  spores,  which  they  closely 
resemble,  except  in  that  they  are  smaller  and  often  have  fewer  cilia. 
In  Ulothrix^  for  example,  and  in  many  of  the  green  algae,  the  gametes 
are,  so  far  as  is  known,  smaller  and  possess  only  two  cilia,  while  the 
larger  asexual  swarm  spores  bear  four  cilia.  Both  sporangia  and 
gametangia  are  homologous  structures,  and,  so  far  as  is  known,  the 
gametes  differ  only  physiologically  from  the  asexual  spores. 

According  to  Strasburger,  to  use  the  language  of  the  translation  '} 

The  sexually  differentiated  plants  manifest  certain  differences  in  their  onto- 
geny, from  which  it  is  possible  to  infer  what  was  the  course  along  which  the 
phylogenetic  differentiation  proceeded  after  sexual  differentiation  had  taken 
place.  The  simplest  case  is  that  in  which  the  product  of  fertilization  gives  rise 
to  an  individual  similar  to  those  which  gave  rise  to  the  product  of  fertilization, 
and  which  closes  its  own  life  history  with  the  development  either  of  sexual 
organs  or  of  asexual  organs  homologous  with  them.  This  occurs  in  many 
Chlorophycece,  where,  from  the  zygospore  (the  product  of  the  coalescence  of 
similar  gametes)  or  the  oospore  (the  product  of  the  coalescence  of  dissimilar 
spermatozoids  and  ova),  a  generation  is  developed  which  resembles  the  preced- 
ing and  gives  rise  either  to  swarm-spores  or  to  sexual  cells  homologous  with 
them.  Generally,  any  one  sexual  generation  follows  after  a  number  of  asexual 
generations,  the  relation  being,  however,  dependent  on  external  conditions,  so 
that,  as  Klebs  has  shown,  the  development  of  a  sexual  or  an  asexual  generation 
can  be  determined  by  the  observer.  In  such  cases  there  is  a  homogeneous 
sequence  of  generations  which  does  not  include  any  other  kind  of  sequence  or 
alternation  beyond  the  development  either  of  asexual  reproductive  organs  or 
of  sexual  organs  homologous  with  them.  The  asexual  reproductive  organs  are 
especially  concerned  with  the  rapid  multiplication  of  individuals  under  favorable 
external  conditions  ;  whilst  sexual  reproduction  is  of  importance  in  maintaining 
the  existence  of  the  species  under  circumstances  which  are  unfavorable  to  the 
vegetative  existence  of  the  individual.  At  the  same  time,  sexual  reproduction 
ensures  certain  advantages  arising  from  the  coalescence  of  distinct  sexual  cells. 

In  proportion  as  the  asexual  mode  of  reproduction  was  replaced  by  the 
sexual,  the  numerical  conditions  of  multiplication  were  maintained  either  by 
the  development  of  a  number  of  oospores,  as  in  certain  Fucaceae ;  or,  in  addi- 
tion to  the  sexual  organs,  altogether  new  organs  were  developed  to  ensure  rapid 
and  vigorous  development  of  new  individuals  in  an  asexual  manner.  This 
took  place  in  various  ways.  Either  asexual  reproductive  organs  were  inter- 
calated in  the  life  history  of  the  original  generation,  or  an  altogether  new 
asexual  generation  was  developed  from  the  product  of  the  sexual  act. 

•  English  translation,  Ann.  Bot.,  8  :  281-316. 


SIGNIFICANCE    OF   THE    SEXUAL    PROCESS.  5 1 

I  have  quoted  thus  at  length  because  it  seems  that  this  statement  of 
Strasburger  is  a  compact  and  concise  summing  up  of  the  phylogenetic 
development  of  the  process  of  reproduction  and  multiplication  of  indi- 
viduals among  the  lower  plants. 

The  intercalation  of  new  asexual  reproductive  organs  into  the  origi- 
nal generation  is  strikingly  illustrated  in  many  of  the  fungi,  in  which 
the  independent  individualization  of  the  different  stages  of  development 
of  the  sexual  generation  into  special  organs  of  vegetative  multiplica- 
tion, or  even  into  distinct  individuals,  was  carried  so  far  that  sexuality 
seems  to  have  disappeared  entirely,  as  in  the  higher  fungi.  On  the 
other  hand,  in  all  plants  beyond  and  including  the  Bryophyta  there 
arose  an  altogether  new  generation  as  the  product  of  the  sexual  act, 
whose  function  is  to  produce  asexually  a  large  number  of  individuals. 
The  degree  of  development  attained  by  the  new  generation  in  the 
plants  above  the  Thallophyta  differs  according  to  whether  its  activity 
was  limited  to  the  production  of  asexual  spores  alone,  or  included 
nutritive  functions  as  well,  or  whether  it  became  an  independent  indi- 
vidual. In  the  Bryophyta^  especially  in  some  of  the  simpler  liver- 
worts, the  new  asexual  generation  is  confined  almost  exclusively  to  the 
production  of  spores,  /.  e.^  to  the  multiplication  of  the  individual, 
while  the  original  or  sexual  generation  upon  which  all  nutritive  func- 
tion is  devolved,  together  with  vegetative  multiplication  as  well,  has 
attained  in  many  cases  a  cormophytic  differentiation.  In  the  Pteri- 
dophyta  and  in  the  higher  plants,  on  the  contrary,  the  center  of  gravity 
of  phylogenetic  evolution  is  transferred  to  the  new  or  asexual  genera- 
tion arising  from  the  act  of  fecundation,  and  in  these  plants  the  asexual 
generation  has  attained  its  highest  cormophytic  development.  Among 
the  Pteridophyta  of  the  present  time  it  is  evident  that  (1.  c,  p.  283) 
'*as  this  evolution  took  place,  the  nutritive  apparatus  of  the  sexual 
generation  became  of  less  importance,  and  it  became  altogether  super- 
fluous from  the  moment  when  the  asexual  generation  began  to  provide 
its  spores  with  the  material  necessary  for  the  development  of  the  sexual 
generation."  Along  with  this  evolution  there  came  into  existence,  as 
a  correlative  phylogenetic  process,  the  dimorphic  character  of  the 
gametophyte,  which  is  characteristic  of  certain  Pteridophyta  and  of 
all  Spermatophyta.  This  dimorphism  was  probably  manifested  in  the 
chaiacter  of  the  mature  gametophyte  before  any  visible  trace  of  it  could 
be  recognized  in  the  unicellular  stage  of  the  sexual  generation,  namely, 
the  spore.  To  illustrate  this  fact  we  need  only  to  recall  the  condition 
which  obtains  among  certain  homosporous  Pilicinece^  for  example, 
Onoclea  struthiopteris  of  the  Polypodiacece.  Here  there  is  no  visi- 
ble evidence  of  heterospory,  yet  it  is  perfectly  well  known  that  in  every 


52  INTRODUCTION. 

culture  of  spores  some  will  develop  into  distinctively  male  prothallia, 
bearing  only  antheridia,  while  others  show  a  marked  tendency  to 
develop  into  prothallia  bearing  only  archegonia.  It  is  also  well 
known  that  this  tendency  toward  dimorphism  is,  in  a  measure,  influ- 
enced by  external  conditions,  for  if  spores  of  Onoclea  struthiopteris 
be  sown  thickly,  and  the  culture  be  poorly  illuminated  and,  conse- 
quently, poorly  nourished,  the  vast  majority  of  the  prothallia  will  be 
male;  but  if  the  spores  be  sown  thinly  and  well  illuminated,  a  much 
greater  number  will  become  female  plants. 

In  all  existing  forms  in  which  the  spores,  or  unicellular  condition 
of  the  sexual  generation,  contain  food  material  for  the  development  of 
the  asexual  generation,  or  its  earlier  stages,  dimorphism  is  well  estab- 
lished, i.  e.,  those  forms  are  heterosporous,  and  the  conclusion  which 
most  naturally  follows  is  that  heterospory  and  the  disappearance  of 
the  nutritive  apparatus  of  the  sexual  generation  represent  correlative 
phylogenetic  processes. 

Now,  during  this  phylogenetic  evolution  and,  as  Strasburger  very 
clearly  puts  it, — 

In  accordance  with  the  general  law  which  determines  the  phylogenetic  disap- 
pearance of  organs  which  have  become  useless,  the  vegetative  parts  of  the  sexual 
generation  became  more  and  more  reduced,  until  little  was  left  but  the  repro- 
ductive organs  themselves :  hence  the  progressive  reduction  in  the  prothallium 
from  the  Ferns  up  to  the  Phanerogams.  This  reduction  culminated  in  the 
complete  loss  of  independent  existence  by  the  sexual  generation,  because  it  had 
ceased  to  be  able  to  nourish  itself  independently,  and  [because  of]  its  becoming 
enclosed  by  the  asexual  generation.  In  consequence  of  this  enclosure  of  the 
sexual  in  the  asexual  generation,  the  advantageous  rapid  multiplication  of  indi- 
viduals which  the  latter  originally  effected  was  lost :  in  order  to  compensate  for 
this  loss,  a  large  number  of  seeds  were  produced  in  the  Phanerogams  in  place 
of  the  numerous  spores  of  the  Cryptogams ;  that  is,  multiplication  is  effected 
now  by  the  product  of  fertilization  instead  of  by  asexual  spores. 

In  harmony  with  this  doctrine,  an  alternation  of  generations  is  neces- 
sary in  those  plants  in  which  the  fecundated  egg  gives  rise  to  the 
asexual  generation,  and  the  asexual  spore  to  the  sexual  generation. 

The  development  of  the  plant  kingdom,  at  least  so  far  as  sexuality 
is  concerned,  seems  to  show  that  sexual  differentiation  was  preceded 
by  asexuality,  and  in  those  groups  in  which  a  true  alternation  of  gen- 
erations exists  the  sexual  generation  is  to  be  regarded  as  the  older 
and  more  primitive  and  as  having  arisen  from  an  asexual  form.  In 
fact,  we  are  able  to  trace  this  phylogenetic  development  step  by  step, 
or  the  evidence  at  hand,  at  least,  seems  to  be  sufficiently  conclusive  to 
justify  the  general  acceptance  of  the  doctrine.  Probably  the  first  indi- 
cation of  this  development  is  to  be  found  among  such  algae  as  CEdo- 


SIGNIFICANCE    OF    THE    SEXUAL    PROCESS.  53 

gonium^  Coleochcete  and,  as  the  researches  of  Oltmanns  seem  to 
indicate  (See  Chapter  IV),  certain  Rhodophycex.  From  the  fecun- 
dated egg  of  CEdogonium  four  swarm-spores  are  developed,  while  in 
Coleochcete  a  multicellular  body  is  developed,  from  the  cells  of  which 
asexual  swarm-spores  are  formed.  In  both  cases  the  swarm-spores 
give  rise  to  sexual  plants,  or  the  first  generation.  The  product  of  the 
fecundated  egg  in  Coleochcete  bears  a  striking  resemblance  to  the 
sporophyte  of  such  liverworts  as  Riccia.  The  fundamental  differ- 
ences lie  chiefly  in  the  fact  that  the  covering  of  the  sporophyte  in 
Coleochcete  is  derived  from  vegetative  branches  of  the  thallus,  the 
oogonium  being  unicellular,  and  that  the  asexual  spores  are  motile,  a 
correlation  with  the  aquatic  habit  of  Coleochcete.  In  the  Rhodo- 
phycece  the  cystocarp  or  cystocarps  are  the  product  of  the  fecundated 
egg,  and  the  spores  give  rise  to  the  first  generation.  This  is  made  all 
the  more  probable  by  the  researches  of  Oltmanns,  which  go  to  show 
that  the  fusion  of  the  cells  of  the  sporogenous  filaments  with  auxiliary 
cells  is  merely  a  nutritive  process.  It  is  of  interest  to  note  further 
that  a  similar  condition  is  preserved  in  certain  Ascomycetes  in  which 
Harper  has  proved  that  unquestioned  sexuality  exists.  Such  algse 
as  Coleochcete.,  therefore,  seem  to  point  out  more  or  less  clearly  the 
phylogenetic  road  along  which  the  ancestors  of  the  Archegoniates  have 
passed. 

Research  upon  the  process  of  fecundation  and  indirect  nuclear 
division,  especially  in  reproductive  cells,  during  the  past  twenty  years, 
has  given  a  new  insight  into  the  significance  of  sexuality  and  the  alter- 
nation of  generations  in  plants.  Our  knowledge  along  this  line  was 
very  materially  advanced  by  the  discovery  of  Van  Beneden  ('83)  that 
the  number  of  chromosomes  is  the  same  in  both  conjugating  nuclei. 
Further  investigations  have  established  the  still  more  important  fact 
that,  in  both  plants  and  animals,  a  reduction  to  one-half  of  the  number 
of  chromosomes  in  the  sexual  nuclei  preceded  the  sexual  act,  and  that, 
as  a  consequence  of  the  fusion  of  the  male  and  female  nuclei,  the 
number  of  chromosomes  in  the  fecundated  egg  is  doubled. 

In  all  the  higher  plants  it  is  a  well-established  fact  that  the  numeri- 
cal reduction  of  the  chromosomes  takes  place  in  the  spore  mother-cell, 
and  that  in  the  cells  of  the  gametophyte  arising  from  the  spore  the 
reduced  number  persists.  In  cells  of  the  sporophyte,  resulting  from 
the  fecundated  ^%^,',  the  increased  number  obtains  until  the  differentia- 
tion of  the  spore  mother-cells.  It  will  thus  be  seen  that  the  funda- 
mental characteristic  of  both  sexual  and  asexual  generations  lies  in  the 
number  of  the  chromosomes,  and  upon  this  phenomenon  rests  the 
sexual  differentiation  of  cells. 


54  INTRODUCTION. 

There  is  a  possibility  that  this  doctrine  may  not  be  applicable  to 
cases  of  apogamy,  apospory,  and  normal  parthenogenesis  among 
plants.  It  has  been  suggested  by  Strasburger  ('94,  p.  300)  that  the 
number  of  chromosomes  may  become  doubled  under  the  influence  of 
correlative  processes  in  an  apogamously  developed  fern  which  arises 
as  a  bud  from  the  prothallium,  the  nuclei  of  vv^hose  cells  contain  the 
reduced  number,  and  for  the  same  reason  the  reverse  may  take  place 
in  cases  of  apospory,  i.  e.,  the  aposporous  development  of  prothallia 
may  be  attended  vv^ith  a  correlative  reduction  in  the  number  of  chromo- 
somes. Until  the  facts  are  determined  by  actual  observation,  all 
discussion  of  this  subject  must  remain  a  matter  of  pure  speculation. 

The  researches  of  Juel  (1900)  upon  the  normal  parthenogenesis  of 
Antennaria  alpina  are  of  the  highest  interest  in  this  connection,  as 
they  throw  light  upon  this  question  so  far,  at  least,  as  the  seed-bearing 
plants  are  concerned.  In  Antennaria  alpina^  in  which  the  egg 
develops  parthenogenetically  under  normal  conditions,  Juel  finds  that 
no  reduction  in  the  number  of  chromosomes  takes  place  in  the  develop- 
ment of  the  embryo-sac,  and,  consequently,  the  nucleus  of  the  egg- 
cell  which  gives  rise  to  the  parthenogenetic  embryo  contains  the  same 
number  of  chromosomes  as  the  vegetative  cells.  Contrary  to  Anten- 
naria dioica^  in  which  fecundation  regularly  occurs,  the  mother-cell 
of  the  embryo-sac  of  A.  alpina  develops  immediately  into  the  embryo- 
sac,  the  heterotypic  and  homotypic  nuclear  divisions  which  follow  the 
appearance  of  the  reduced  number  of  chromosomes  being  omitted. 

In  cases  of  normal  parthenogenesis  among  the  angiosperms,  the 
facts,  so  far  as  they  are  known,  are  certainly  not  at  variance  with  the 
doctrine  of  the  reduction  of  the  chromosomes  as  applied  to  the  alter- 
nation of  generations. 

As  has  been  intimated  in  preceding  paragraphs,  the  sexual  genera- 
tion has  been  spoken  of  as  the  more  primitive  condition,  and,  as  will 
be  seen  from  the  following,  the  reduction  in  the  number  of  chromo- 
somes in  the  spore  mothei--cell  is  regarded  by  Strasburger  as  the 
return  of  highly  organized  plants  to  the  original  unicellular  condition  : 

The  morphological  cause  of  the  reduction  in  the  number  of  chromosomes 
and  of  their  equality  in  number  in  the  sexual  cells  is,  in  my  opinion,  phylo- 
genetic.  I  look  upon  these  facts  as  indicating  a  return  to  the  original  generation 
from  which,  after  it  had  attained  sexual  differentiation,  off"spring  was  developed 
having  a  double  number  of  chromosomes.  Thus  the  reduction  by  one-half  of 
the  number  of  the  chromosomes  in  the  sexual  cells  is  not  the  outcome  of  a 
gradually  evolved  process  of  reduction,  but  rather  it  is  the  reappearance  of 
the  primitive  number  of  chromosomes  as  it  existed  in  the  nuclei  of  the  genera- 
tion in  which  sexual  diffierentiation  first  took  place  (1.  c,  p.  288). 

The  phenomenon  under  consideration  is  essentially  that  of  the  return  of  the 


SIGNIFICANCE    OF   THE    SEXUAL   PROCESS.  55 

most  highly  organized  plants,  at  the  close  of  their  life-cycle,  to  the  unicellular 
condition :  in  a  word  it  is  the  repetition  of  phylogeny  in  ontogeny  (1.  c, 
P-3"). 

This  theory  of  reduction  must  still  be  regarded  as  a  very  helpful 
working  hypothesis,  finding  its  greatest  application  in  the  higher 
plants.  In  the  lower  cryptogams  the  theory  is  confronted  with  facts, 
many  of  which  seem  at  present  to  be  quite  at  variance  with  it.  The 
product  of  fecundation  in  the  Thallophyta  as  a  rule  does  not  give  rise 
to  a  definite  organism  representing  the  asexual  generation,  and  it  is  not 
known  at  which  point  in  the  life-cycle  that  reduction  takes  place.  It 
has  been  suggested  that  reduction  may  take  place  during  the  germina- 
tion of  the  zygote  or  oospore.  Conclusions  respecting  the  time  of 
reduction  in  the  lower  cryptogams  have  been  drawn  chiefly  from  the 
phenomena  of  certain  cell-divisions  that  seem  to  be  analogous  with 
divisions  which  follow  the  reduction  in  higher  organisms,  and  not 
from  an  actual  determination  of  the  number  of  chromosomes.  On 
account  of  the  many  difficulties  in  counting,  the  number  of  chromo- 
somes is  known  in  only  a  very  few  algae  and  fungi,  and  our  knowledge 
on  this  subject  is  so  meager  with  respect  to  these  plants  that  the  few 
definite  facts  that  have  been  obtained,  although  apparently  at  variance 
with  the  theory,  may  not  as  yet  be  considered  as  offering  very  serious 
objections  to  it. 

If  the  reduction  in  the  number  of  chromosomes  signifies  what  is 
attributed  to  it  by  the  theory,  it  is  possible,  in  the  light  of  facts  that 
have  been  observed  in  such  algae  as  Fucus  and  Dictyota^  that  what  is 
considered  the  sexual  generation  in  the  Thallophyta  may  not  be 
homologous  with  the  gametophyte  of  higher  plants,  assuming  that 
homology  is  based  upon  the  number  of  chromosomes.  Farmer  and 
Williams  ('96,  '98),  and  Strasburger  ('97)  have  found  that  the  reduced 
number  of  chromosomes  in  Fucus  appears  in  the  oogonium,  while  in 
vegetative  cells  of  the*  thallus  twice  that  number  is  present.  Stras- 
burger finds  that  in  the  first  nuclear  division  in  the  oogonium  the 
reduced  number  appears,  fourteen  to  sixteen  having  been  counted, 
and  this  number  persists  throughout  the  two  succeeding  mitoses.  In 
vegetative  cells  of  the  thallus,  which  is  regarded  as  the  gametophyte, 
the  number  is  not  far  from  thirty.  In  Dictyota  I  have  found  the 
reduced  number  (sixteen)  of  chromosomes  in  the  first  nuclear  division 
of  the  tetraspore  mother-cell,  while  in  the  vegetative  cells  of  the  thallus 
bearing  the  tetrasporangia  about  twice  that  number  was  counted. 
Whether  in  the  nuclei  of  plants  arising  from  tetraspores  the  reduced 


^6  INTRODUCTION. 

number  persists,  and  whether  in  the  egg-cell  this  number  obtains  was 
not  determined.^ 

As  is  well  known,  two  views  are  held  concerning  the  manner  in 
which  the  reduction  in  the  number  of  chromosomes  is  accomplished. 
One  of  these  views,  which  has  been  given  prominence  by  Weismann, 
holds  that  the  chromosomes  are  qualitatively  different,  and  that  reduc- 
tion is  accomplished  during  the  maturation  divisions  in  animal  cells 
and  in  the  first  two  divisions  taking  place  in  the  spore  mother-cells  of 
higher  plants.  For  example,  in  the  second  maturation  division  of  the 
animal  egg  it  is  maintained  that  the  daughter  chromosomes  do  not  arise 
as  a  result  of  a  longitudinal  splitting,  but  by  a  transverse  division,  or 
what  is  known  as  a  qualitative  division.  The  nuclei  of  the  four  cells 
thus  resulting,  whether  representing  the  egg  and  its  polar  bodies  or 
those  which  develop  directly  into  spermatozoa,  are  hereditarily  different 
in  character,  and  it  is  upon  this  assumption  that  hereditary  variation  is 
based. 

The  other  view,  which  is  now  very  generally  accepted  by  botanists, 
is  that,  in  plants  no  qualitative  division  exists,  but  the  chromosomes 
of  each  mitosis  arise  in  every  case  by  a  longitudinal  splitting.  The 
reduction  takes  place  in  the  resting  nucleus  or  during  the  early  pro- 
phase of  the  first,  or  heterotypic,  mitosis  in  the  spore  mother-cell  of 
higher  plants.  The  fact,  as  shown  in  preceding  paragraphs,  that 
during  this  first  mitosis  a  double  longitudinal  splitting  of  the  chromo- 
somes occurs,  probably  as  a  preparation  for  the  two  divisions,  has  led 
to  much  confusion,  because  these  divisions  were  supposed  to  have  been 
rather  the  instrument  of  reduction  than  a  consequence  of  reduction. 

Assuming  the  persistent  individuality  of  the  chromosomes,  we  may 
conclude  on  good  grounds  that  the  reduction  represents  the  actual  and 
complete  fusion  of  the  chromosomes  of  both  parents,  which  have 
remained  separate  in  the  sporophyte  until  the  formation  of  the  spore 
mother-cells.  There  is  no  visible  evidence  that  a  qualitative  difference 
exists  between  the  chromosomes  in  plants,  and  our  assumption  here  is 
that  they  are  hereditarily  similar,  because  of  the  fact  that  every  indi- 
rect nuclear  division  is  preceded  by  a  longitudinal  splitting  of  the 
chromatin. 

Since  the  nucleus  is  the  unquestionable  bearer  of  hereditary  char- 
acters, fusion  of  sexual  nuclei  in  fecundation  has  for  its  purpose 
the  blending  of  two  lines  of    descent    and   possibly  the    restoration 

'J.  Lloyd  Williams  in  a  recent  paper  (Studies  in  the  Dictyotacese,  Ann.  Bot.,i8:  141-160,  1904) 
observes  facts  that  seem  to  point  to  the  conclusion  that  the  plantlets  developing  from  the  tetraspores, 
with  their  reduced  number  of  chromosomes,  may  become  gametophytes,  and  that  the  fecundated  egg 
cells  probably  develop  into  tetraspore  plants  which  have  been  shown  to  possess  the  increased  number 
of  chromosomes.     If  this  be  true,  an  alternation  of  generations  exists  in  Dictyota. 


SIGNIFICANCE    OF   THE    SEXUAL    PROCESS.  57 

of  the  power  of  growth  and  cell-division.  The  influence  of  the 
hereditary  characters  of  each  parent  upon  each  other  by  their  intimate 
association  in  the  same  nucleus  seems  to  be  the  physical  basis  of 
phylogenetic  variation,  but  the  manner  in  which  this  influence  acts  to 
bring  about  variation,  or  to  impart  a  more  vigorous  character  to  the 
product  of  fecundation  still  remains  a  matter  of  speculation. 

It  is  well  to  consider  the  blending  of  the  two  lines  of  descent  as  a 
consequence  of  fecundation  in  a  relative  sense  or  as  a  correlative 
phylogenetic  process.  In  certain  of  the  lower  cryptogams,  Ulothrix 
and  Basidiobolus  for  example,  in  which  the  gametes  arise  from 
adjacent  cells  of  the  same  filament  and  in  which  a  sexual  differentia- 
tion is  not  at  all  or  only  scarcely  recognizable,  there  does  not  seem  to 
be  two  lines  of  descent  to  blend,  yet  it  is  conceivable  that  the  sexual 
character  of  the  nuclei  may  have  been  determined  before  the  stage  of 
ontogeny  is  reached  in  which  the  sexual  cells  manifest  themselves  as 
such.  If  in  such  forms  a  reduction  in  the  number  of  chromosomes 
occurs,  the  sexual  character  of  the  nuclei  is  determined  at  that  time. 
It  is  well  known  that  among  the  simpler  forms  of  the  algse  and  fungi, 
the  development  of  gametes  depends  to  a  certain  extent  upon  external 
conditions,  which  effect  transpiration,  atmospheric  pressure,  food 
supply,  and  so  forth,  yet  no  one  would  suppose  for  one  moment  that 
sexuality  is  the  outcome  of  these  external  conditions. 

We  have  now  to  touch  briefly  upon  the  category  of  phenomena  by 
which  a  growth  stimulus,  or  the  power  of  growth  and  cell-division,  is 
imparted  to  the  product  of  fecundation.  Among  many  of  the  lower 
algae  about  the  only  important  difference  which  seems  to  exist  between 
a  gamete  and  an  asexual  swarm-spore  is  the  ability  of  the  latter  to 
develop  into  a  normal  individual  of  the  adult  size.  It  is  true  that  the 
iso-gametes  of  algae,  such  as  Ulothrix.,  are  capable  of  developing  into 
small  dwarf  individuals — a  fact  which  indicates  that  here,  at  least,  the 
gametes  possess  the  power  of  independent  growth  sufficiently  to  enable 
the  resulting  plantlet  to  develop  to  a  limited  extent.  As  soon,  how- 
ever, as  the  sexual  elements  have  attained  any  marked  degree  of 
bisexual  differentiation  in  the  plant  kingdom,  the  individual  gametes 
are  quite  incapable  of  independent  development  even  into  the  most 
rudimentary  individuals,  cases  of  normal  and  artificial  parthenogenesis 
excepted. 

The  stimulus  to  growth  and  division  in  bisexual  reproductive  cells 
is  imparted  normally  only  by  the  fusion  of  male  and  female  elements, 
and  the  question  naturally  arises,  is  this  stimulus  due  to  the  fusion  of 
the  cytoplasm  of  the  male  cell  with  that  of  the  female,  or  is  it  due 
merely  to  the  fusion  of  the  respective  nuclei  ?     Experiments  upon  arti- 


58  INTRODUCTION. 

ficial  parthenogenesis,  brought  about  by  the  use  of  chemicals  and  other 
stimuli,  have  thrown  some  light  upon  the  subject,  but  in  the  opinion 
of  the  author  they  are,  as  yet,  far  from  furnishing  an  adequate  solution 
of  the  problem. 

In  Marsilia  vestita  Nathansohn  (1900)  fovmd  that  it  was  possible 
to  stimulate  the  egg-cell  to  a  parthenogenetic  development  by  exposing 
the  germinating  macrospores  to  a  temperature  of  35°  C.  for  24  hours, 
and  allowing  them  to  continue  their  development  at  a  temperature  of 
27°  C.  As  a  result  about  7  per  cent,  of  the  spores  gave  rise  to  par- 
thenogenetic embryos.  So  far  as  we  know,  this  is  the  only  case  among 
plants  above  the  Thallophyta  in  which  parthenogenesis  has  been 
brought  about  artificially,  and  it  may  be  that  Marsilia  lends  itself  to 
this  sort  of  experiment  more  readily  because  of  the  fact  that  in  certain 
species  the  tendency  toward  normal  parthenogenesis  is  strongly  mani- 
fested. In  Marsilia  drummondii  Shaw  ('97)  found  normal  parthe- 
nogenesis to  be  of  frequent  occurrence.  In  these  cases  of  Marsilia 
the  morphological  side  of  the  question,  especially  the  behavior  of  the 
nucleus,  is  not  known,  nor  have  the  number  of  chromosomes  been 
determined  in  the  cells  of  the  parthenogenetic  embryo. 

On  the  animal  side  of  the  question  the  experimenter  finds,  fortu- 
nately, an  abundance  of  most  favorable  material  in  the  eggs  of  sea- 
urchins  and  of  certain  marine  worms.  The  results  of  several  investi- 
gators (Wilson,  Morgan,  Loeb,  and  others)  have  shown  that  the  eggs 
of  Arbacia  and  Toxopenustes  may  be  made  to  develop  parthenoge- 
netically  through  certain  earlier  stages  by  subjecting  them  for  a  certain 
time  to  a  solution  of  sea-water,  whose  osmotic  power  is  increased  by 
the  addition  of  a  solution  of  magnesium  chloride.  The  action  of  the 
Mg-solution  seems  to  be  similar  to  the  growth  stimulus  imparted  to 
the  egg  by  a  spermatozoon  in  normal  fecundation. 

Equally  instructive  are  the  experiments  of  Winkler  (1901)  on  nucle- 
ated and  enucleated  fragments  of  the  egg  of  Cystosira  barbata,  one 
of  the  Fucacece.^  which  were  fecundated  by  the  spermatozoids.  Both 
the  enucleated  fragments  and  those  containing  the  nuclei  developed 
into  small  embryo  plantlets  which  were  exactly  alike  and  attained 
about  the  same  stage  of  development. 

The  development  of  normally  fecundated  fragments  of  egg-cells  and 
that  of  the  entire  eggs  induced  to  develop  parthenogenetically  by 
chemical  or  physical  stimuli  are  phenomena  which  seem  to  fall  into 
the  same  category.  They  show  that  in  all  probability  the  growth 
stinaulus,  or  the  restoration  of  the  power  of  division  and  the  blending 
of  hereditary  characters  are  phenomena  which  in  a  measure  are  inde- 
pendent of  each  other.     Experiments  similar  to  the  foregoing  have 


SIGNIFICANCE    OF    THE    SEXUAL    PROCESS.  59 

their  greatest  value  in  the  suggestiveness  of  their  results  and  the  new 
points  of  view  to  which  these  results  lead.  They  do  not  show  that 
the  reactions  brought  about  by  these  stimuli  are  the  same  as  those 
resulting  from  the  union  of  sexual  cells.  Although  the  development 
of  a  rudimentary  embryo  induced  by  artificial  means  may  proceed  in 
the  same  manner  as  the  product  of  normal  fecundation,  yet  the  arti- 
ficial stimulus  cannot  be  looked  upon  as  being  equivalent  to  the  sexual 
process.  In  the  case  of  the  former,  we  are  dealing  with  a  stimulus 
which  merely  starts  growth,  but  a  mature  individual  is  never  developed. 
The  sting  of  an  insect  or  some  similar  stimulus  may  call  forth  a 
growth  in  a  leaf  of  an  oak,  which  results  in  a  gall,  a  local  and  limited 
growth,  but  never  in  an  oak  tree,  and  we  cannot  for  one  moment 
think  of  comparing  such  a  stimulus  to  a  sexual  process. 

The  author  does  not  agree  with  those  who  regard  the  sexual  process 
merely  as  a  restoration  to  the  egg  of  the  power  of  growth  and  division. 
We  are  not  quite  ready  to  lay  aside,  as  yet,  the  facts  won  by  twenty 
years  of  the  most  careful  morphological  research  for  any  chemical  or 
electrical  theory  of  heredity. 

Our  knowledge  of  sexual  reproduction  in  the  plant  kingdom  indi- 
cates beyond  question  that  that  which  is  of  primary  significance  in  the 
sexual  process  is  the  fusion  of  the  nuclei,  and  the  question  still 
remains,  which  imparts  the  growth  stimulus,  the  nucleus  or  the  cjrto- 
plasm  of  the  sperm  ?     Or  are  both  necessary  ? 

Strasburger  has  suggested  that  the  stimulus  to  grov/th  and  division 
is  given  by  the  cytoplasm,  and  especially  a  particular  part  of  the  same, 
the  kinoplasm,  brought  into  the  egg  by  the  spermatozoid.  Some 
zoologists  have  attributed  this  stimulus  to  the  centrosome  of  the  sperm, 
but  in  the  plant  kingdom  no  case  is  definitely  known  in  which  a 
centrosome  is  brought  into  the  egg  by  a  spermatozoid.  The  doctrine 
of  Strasburger  is  perhaps  the  best  that  has  been  proposed,  and  it  seems 
to  have  some  basis  in  fact.  According  to  this  view  the  egg  is  rich 
in  food  material,  trophoplasm,  and  poor  in  kinoplasm,  while  in  the 
sperm  the  reverse  obtains.  The  unfecundated  egg  is  incapable  of 
developing,  therefore,  on  account  of  the  lack  of  energy. 

This  theory,  however  plausible  it  may  seem,  leaves  much  to  be 
desired.  In  the  first  place,  it  is  not  known  as  a  fact  that  the  egg  is 
poor  in  kinoplasm,  and  that  the  sperm  is  correspondingly  rich  in  that 
substance.  In  many  cases  the  quantity  of  cytoplasm  of  the  male  cell 
is  so  small  that  it  seems  almost  incredible  that  it  could  have  such  a 
powerful  influence.  The  spermatozoid  of  the  fern,  for  example,  con- 
sists of  a  relatively  very  small  amount  of  cytoplasm,  and  the  kino- 
plasmic  part  of  this  constitutes  an  organ  of  locomotion.     Although 


6o  INTRODUCTION. 

cytoplasmic  band  and  blepharoplast,  or  cilia-bearer,  enter  the  egg, 
yet  their  function  seems  to  be  of  secondary  importance  as  compared 
with  that  of  the  nucleus.  Again  in  the  higher  seed-bearing  plants, 
the  generative  nuclei  are  accompanied  by  only  a  small  portion  of  cyto- 
plasm, which  cannot  be  recognized  in  the  embryo-sac,  and  it  seems 
reasonable  that  it  is  merely  absorbed  as  so  much  food.  However, 
when  we  remember  that  in  all  cases  of  fecundation  at  least  some 
cytoplasm  accompanies  the  male  nucleus  into  the  egg,  there  is  good 
ground  for  the  belief  that  the  cytoplasm  plays  some  important  r61e, 
but  whether  that  be  anything  more  than  to  assist  in  restoring  the 
power  of  growth  and  division  must  at  present  remain  a  question. 

The  behavior  of  the  sexual  nuclei  during  the  process  of  fecundation 
and  the  wonderful  phenomena  of  karyokinesis  point  to  the  conclusion 
that  the  nucleus  is  the  bearer  of  hereditary  characters,  and  that  the 
blending  of  these  characters  in  the  offspring  are  largely  the  result  of 
the  fusion  of  the  sexual  nuclei.  The  nuclear  fusion  is  also  the  basis 
of  all  hereditary  variation. 


CHAPTER  II.— FECUNDATION;    MOTILE  ISO- 
GAMETES. 

ULOTHRIX  AND  HYDRODICTYON. 

There  seems  to  be  no  question  that  the  simplest  and  most  primitive 
form  of  sexuality  consists  in  the  union  of  motile  isogametes  as  found 
among  many  of  the  most  primitive  algae.  The  chief  difference  be- 
tween the  gametes  of  such  forms  as  Pandorina  and  Ulothrix^  for 
example,  and  their  asexual  swarm-spores,  from  which  the  gametes 
were  undoubtedly  derived  phylogenetically,  seems  to  be  merely  phys- 
iological. Generally  speaking,  the  gamete  is  incapable  of  developing 
into  a  normal  adult  individual.  It  must  unite  first  with  another  gamete 
of  the  same  species  in  order  to  restore  the  power  of  growth  and  divis- 
ion necessary  to  the  development  into  an  individual  common  to  the 
species,  and  apart  from  theoretical  considerations  (I  refer  to  the  num- 
ber of  chromosomes  which,  of  course,  has  not  been  determined  for 
these  lower  forms)  this  is  the  most  fundamental  distinction  made. 
Many  other  well-known  forms  among  the  green  algae  might  have  been 
taken  as  representatives,  instead  of  the  two  selected,  but  these  have 
been  chosen  because  the  development  of  the  reproductive  cells  from 
the  mother-cell  has  been  more  carefully  worked  out  here,  and  because 
the  processes  in  this  development  are  coming  to  be  regarded  as  more 
important  from  a  genetic  standpoint. 

In  connection  with  Ulothrix  I  have  selected  Hydrodictyon  in  order 
to  present  the  cytological  processes  preparatory  to  the  formation  of 
gametes  in  uninucleate  as  well  as  in  multinucleated  cells. 

The  cytological  development,  leading  to  the  formation  of  gametes 
and  also  asexual  swarm-spores  among  the  simpler  representatives  of 
the  green  algae,  has  been  investigated  by  a  number  of  earlier  observers, 
among  whom  were  Alexander  Brown,  Cohn,  Pringsheim,  Dodel, 
Strasburger,  Klebs,  and  lately  by  Timberlake. 

The  well-known  and  widely  distributed  Ulothrix  consists  of  a  simple 
unbranched  filament  differentiated  into  base  and  apex  (Fig.  17,  A). 
The  cells,  except  the  basal  one,  which  is  modified  as  an  organ  of 
attachment,  are  quite  alike.  Each  contains  a  single  nucleus  and  a 
band-shaped  chloroplast  in  the  form  of  an  almost  complete  hollow 
cylinder.  Almost  any  vegetative  cell  of  the  filament  save  the  basal 
one  may,  without  undergoing  any  external  modification,  function  as  a 
gametangium. 


62 


FECUNDATION  ;     MOTILE    ISOGAMETES. 


The  process  of  cell-formation  by  which  the  gametes  are  devel- 
oped from  the  protoplast  of  the  gametangium  has  been  observed  and 
described  in  some  detail  by  Dodel  ('76)  and  by  Strasburger  ('92). 
These  authors  agree  that  the  gametes  arise  not  by  the  process  of  free 
cell-formation,  as  understood  at  the  time,  but  by  successive  bipartitions 
of  the  entire  plasmic  contents  of  the  cell.  According  to  Strasburger 
('92)  the  process  of  division  in  the  development  of  the  swarm-spores, 
which  is  exactly  the  same  for  the  gametes,  differs  from  the  beginning 
in  a  very  marked  way  from  the  vegetative  cell-divisions.  At  first  the 
cell-contents  undergo  apparently  a  sort  of  rejuvenescence  by  which 
the  protoplast  becomes  independent  of  both  the  outer  and  inner  plasm 

D 


Fig.  17. — Ulothrix  zonata. — (After  Dodel-Port.) 

A,  young  plant.     B,  Zoosporangia,  showing  escape  of  swarm-spores. 

C,  an  asexual  swarm-spore.     D,  gametangia,  showing  gametes  and  escape  of  same. 

E,  two  gametes.     F,  G,  copulation  of  gametes. 

H,  zygote.    J,  zygote  after  a  period  of  rest. 

K,  germinating  zygote  whose  contents  have  divided  into  a  number  of  swarm-spores. 


membranes.  In  the  first  division  the  granular  plasma  only  is  halved. 
The  outer  plasma  membrane  (Hautschicht)  is  undivided,  and  the 
membrane  surrounding  the  vacuole  remains  unchanged.  By  further 
successive  divisions  these  two  protoplasts  give  rise  ultimately  to  the 
gametes.  The  process  of  division  is  the  same  whether  gametes  or 
asexual  swarm-spores  result  (Fig.  17,  D).  Strasburger  has  expressed 
the  opinion  that,  in  the  development  of  the  gametes,  only  one  more 
cell-division  is  necessary  above  those  required  for  the  zoospores,  and 
this  division  renders  the  resulting  cells  or  gametes  incapable  of  further 
independent  development.  In  what  way  this  last  division  incapaci- 
tates the  gametes  for  further  independent  development  was  not  dis- 
cussed at  the  time.     That  view  was  probably  prompted  by  Weismann's 


ULOTHRIX  AND  HYDRODICTYON.  63 

theory  of  a  reduction  division  of  the  chromosomes,  which  at  the  time 
received  a  wider  acceptance  than  at  present. 

In  Hydrodictyon  Klebs  ('91)  affirms  that  the  process  of  cell-forma- 
tion, giving  rise  to  gametes  or  asexual  swarm-spores,  occupies  an 
intermediate  position  between  simultaneous  and  successive  cell-division. 
From  what  follows  it  will  be  seen  that  the  process  is  a  cleavage 
similar  to  that  occurring  in  certain  Phycomycetes,  but,  using  the 
methods  that  he  did,  Klebs  failed  to  perceive  the  true  nature  of  the 
process.     His  account  in  substance  is  as  follows  : 

The  first  indication  of  cleavage  is  manifested  in  the  appearance  of 
numerous  small  clefts,  pointed  at  the  ends,  in  the  plasma  layer  con- 
taining the  chlorophyll  (Fig.  18,  A).     This  can  be  seen  in  material 
cultivated  in  darkness  in  a  maltose  solution,  especially  after  the  appli- 
cation of  a   weak  plasmolysing  agent.     These   clefts   soon   become 
longer  and  more  numerous,   neighboring  ones  thereby  uniting  with 
each  other,  so  that  finally  the  entire  chlorophyll-bearing  layer  is  seg- 
mented into  pieces  which  are  still  connected,  however,  by  fine  plasmic 
threads.     The   cleavage   is    not   confined  solely  to   the  chlorophyll- 
bearing  layer,  but  extends  into  the  colorless  plasma  in  which  the  nuclei 
are  situated.     The  plasma  membrane  and  the  wall  of  the  vacuole  are, 
on  the  contrary,  unaffected.     Previously  to  and  during  the  cleavage 
the  plasmic  layer  concerned  frequently  undergoes  a  contraction,  thus 
giving  rise  to  colorless  spaces,  so  that  this  layer  appears  as  a  coarse 
net,  as  Pringsheim  ('71)  has  described  for  Bryopsis.     These  spaces 
contain  also  some  plasma,  and,  as  the  plasma  membrane  and  wall  of 
the  vacuole  are  continuous,  the  entire  cell  contents  form  still  a  unit, 
as  shown  by  plasmolys^is.     The  continuation  of  the  cleavage  results  in 
the  segmentation  of  the  plasmic  contents  into  numerous  bands  with 
irregular  and  sinuous  contour  (Fig.   18,  B).     These  bands    undergo 
still  further  segmentation  (Fig.  18,  C),  until  finally  the  plasmic  con- 
tents are  broken  up  into  numerous  small  pieces,  each  containing  a 
nucleus,  which  ultimately  separate  and  develop  into  gametes  (Fig.  18, 
D).     The  method  of  division  in  these  portions  referred  to  in  Fig.  18, 
B,  C  (Klebs  continues),  appears  to  consist  in  a  constriction,  progress- 
ing from  one  side,  but  not  entirely  completed,  since  the  individual 
parts  remain  in  communication  ;  yet  direct  observation  shows  also  that, 
in  the  plane  of  division,  a  colorless  line  or  furrow  is  frequently  present, 
which  gives  the  impression  that  the  constriction  may  proceed  from 
within.     The  same  principle  operating  in  the  segmentation  of  the 
bands  or  pieces  obtains  also  in  the  earlier  cleavage  of  the  whole 
plasmic  layer  of  the  cell.     There  is  from  beginning  to  end  a  progres- 
sive condensation,  but  the  process  that  plays  the  chief  r61e  is  concealed 
from  observation. 


64 


fecundation;    motile  isogametes. 


Using  improved  methods  Timberlake  ('oi)  in  a  study  of  spore- 
formation  in  Hydrodictyon  uiriculatum  Roth.,  has  found  that,  in  the 
earlier  stages  of  the  process,  cleavage  takes  place  by  means  of  surface 
constrictions  of  the  plasma  membrane  on  the  outside  and  the  vacuolar 
membrane  on  the  inside  of  the  protoplasmic  layer,  as  may  be  seen  from 
Klebs'  figures  (Fig.  i8,  B,  C).  The  process  is  a  progressive  one,  the 
cleavage  furrows  cutting  out  first  large  irregular  multinucleated  masses 
of  protoplasm,  which  are  in  turn  divided  into  smaller  ones,  until  each 


Fig.  i8.— Cell-cleavage  in  Hydrodictyon  utriculatum.—{Mttt  Klebs.) 

A,  cell  showing  cleavage  furrows  at  early  stage  in  the  process ;  e,  place  in 
protoplasm  free  from  chlorophyll. 

B,  sausage-shaped  protoplasts  formed  in  early  stage  of  cleavage. 

C,  two  protoplasts  similar  to  those  in  B,  showing  manner  of  further  cleavage. 

D,  final  result  of  the  cleavage. 

contains  a  single  nucleus.  In  this  manner  the  entire 
protoplast  is  divided  into  uninucleated  spores  or  gam- 
etes, as  the  case  may  be. 

Judging  from  Strasburger's  account  of  the  process 
in  Ulothrix^  it  seems  probable  that  cell-formation 
leading  to  the  development  of  gametes  or  swarm-spores  is  also  a 
cleavage  similar  to  that  in  Hydrodictyon.  In  Ulothrix,  however, 
the  cells  are  uninucleate,  and  a  nuclear  division  must  either  accompany 
or  precede  cell-division.  Until  the  behavior  of  the  nucleus  is  known, 
and  the  process  carefully  worked  out  with  the  aid  of  more  impi'oved 
methods,  the  exact  nature  of  the  cell-formation  in  question  must 
remain  largely  a  matter  of  conjecture. 

In  the  light  of  more  recent  investigations  concerning  cell-formation 
among  the  lower  thallophytes,  it  is  evident  that  our  present  knowledge 
of  this  process  in  connection  with  the  development  of  gametes  or 
asexual  zoospores  among  the  algae  is  very  meager  and  fragmentary. 


COPULATION    OF    GAMETES. — ECTOCARPUS.  65 

COPULATION  OF  GAMETES. 

The  gametes  of  Ulothrix  zonata  are  rounded  or  oval  cells,  bearing 
two  cilia  at  the  anterior  end  (Fig.  17,  E).  Each  contains  a  nucleus, 
a  red  eye-spot,  situated  about  midway  between  the  ends  of  the  cell 
near  the  surface,  and  a  chromatophore.  According  to  Strasburger 
the  cilia  are  developed  under  the  influence  of  the  nucleus  and  from  the 
anterior,  colorless  portion  or  mouth-piece,  which  consists  mostly  of 
kinoplasm.  In  his  later  investigation  of  the  subject  of  swarm  cells, 
Strasburger  (1900)  finds  that  the  cilia  arise  from  a  local  kinoplasmic 
thickening  of  the  plasma  membrane  at  the  anterior  end.  As  already 
mentioned  in  a  preceding  paragraph  (p.  47),  he  regards  this  thicken- 
ing as  the  homolog  of  the  blepharoplast  of  the  Archegoniates.  In  the 
swarm-spores  of  Hydrodictyon^  Timberlake  finds  a  small  body  at  the 
base  of  the  cilia,  which,  in  some  cases  at  least,  was  not  a  part  of  the 
plasma  membrane. 

The  gametes  copulate  in  pairs  immediately  after  they  escape  from 
the  gametangium  (Fig.  17,  F,  G).  It  is  probable  that  they  may  be 
brought  together,  or  at  least  held  together  after  coming  in  contact,  by 
means  of  a  chemotactic  stimulus.  The  stigmatic  or  eye-spots  do  not 
unite,  but  remain  separate  and  independent  in  the  young  zygote  (Fig. 
17,  H).  There  is  no  doubt  of  a  nuclear  fusion,  but  how  soon  this 
takes  place  after  conjugation  is  not  known,  so  far  as  the  author  is  aware. 

In  Hydrodictyon  the  gametes  are  small,  oval  in  shape,  biciliate, 
containing  one  nucleus  and,  according  to  Klebs,  two  pulsating  vacuoles. 
They  conjugate  in  pairs  immediately  on  escaping  from  the  gametan- 
gium, but  I  have  observed  that  conjugation  may  sometimes  take  place 
within  the  mother-cell.  If,  however,  copulation  does  not  follow  soon 
after  the  gametes  are  set  free,  they  become  incapable  of  uniting,  come 
to  rest  and  disorganize.     Whether  this  is  a  rule  was  not  determined. 

ECTOCARPUS. 

Among  the  isogamous  Phceophycece  the  sexual  process  is  doubtless 
best  known  in  Ectocarpus  siliculosus  Lyngb.  from  the  investigations 
of  Berthold  ('8i),  which  have  been  confirmed  and  extended  by  Oltmanns 
(*99) .  Ectocarpus  is  of  especial  interest  in  this  respect,  since  it  repre- 
sents a  transition  from  isogamy  to  heterogamy.  In  fact,  there  is  in  the 
brown  algae,  as  well  as  in  phylogenetic  series  of  other  Thallophyta^ 
every  transition  from  the  type  of  gametes  found  in  Ectocarpus  to  that 
of  Fucus.  The  gametes,  although  nearly  or  quite  the  same  size  and 
appearing  morphologically  alike,  are  physiologically  different,  and  we 
may,  with  much  propriety,  speak  of  egg-cells  and  spermatozoids. 


66  FECUNDATION  ;     MOTILE    ISOGAMETES. 

Both  Oltmanns  and  Berthold  agree  in  the  opinion  that  Ectocarfus 
siliculosus  may  be  either  monoecious  or  dioecious,  for  they  observed 
individuals  whose  gametes  would  not  conjugate  with  each  other,  but 
only  with  those  of  another  individual.  As  is  well  known,  the  gametes 
are  generally  borne  in  the  so-called  plurilocular  sporangia.  The  details 
in  the  process  of  nuclear  and  cell-division  in  the  development  of  both 
gametes  and  asexual  swarm-spores  have  not,  as  yet,  been  thoroughly 
studied.  The  gametes  (Fig.  19,  A)  are  pear-shaped  cells  with  a  chro- 
matophore,  nucleus,  a  reddish  brown  eye-spot,  and  two  cilia  inserted 
laterally.  The  cilia  are  of  unequal  length,  the  longer  extending  for- 
ward and  the  shorter  backward. 

The  conjugation  of  the  gametes  can  be  most  readily  followed  in  a 
hanging  drop,  into  which  both  male  and  female  gametes  are  intro- 
duced, when  the  whole  process  may  be  observed  with  the  aid  of  the 
highest  magnifying  powers.  The  female  gametes,  as  a  rule,  first  come 
to  rest,  and  about  each  one  numerous  spermatozoids  assemble.  If  the 
female  gamete  comes  to  rest  at  the  edge  of  the  drop,  the  male  cells 
cluster  about  it,  attaching  themselves  apparently  by  the  anterior  cilium, 
giving  the  familiar  picture  figured  by  Berthold  (Fig.  19,  A).  But 
should  the  female  gamete  attach  itself  to  some  particle  hanging  in  the 
arched  surface  of  the  drop,  this  cell  then  appears  as  a  circular  disk 
surrounded  by  a  wreath  of  male  cells  radially  disposed.  Shortly  a 
male  gamete  (in  exceptional  cases  two),  having  attached  itself  to  the 
female  by  means  of  the  anterior  cilium,  approaches  the  latter  appar- 
ently by  the  sudden  contraction  of  the  same  and  unites  with  it,  while 
the  remaining  male  gametes  withdraw  (Fig.  19,  B,  C).  In  a  few 
minutes  cytoplasmic  union  is  complete,  and  within  about  ten  hours 
after  copulation  both  nuclei  have  fused  (Fig.  19,  E,  F,  G).  The 
chloroplasts  do  not  unite,  a  fact  which  is  contrary  to  the  peculiar 
phenomenon  described  by  Overton  for  Sfirogyra  (see  page  69). 

The  sexual  process  in  Ulothrix^  Hydrodtctyon,  and  Ectocarpus 
may  be  considered  as  fairly  typical  of  the  lower  algae  in  which  fecun- 
dation consists  in  the  fusion  of  motile  isogametes.  In  this,  probably 
the  simplest  and  most  primitive  sexual  process,  as  in  the  higher  plants, 
it  will  be  seen  that  fecundation  consists  in  the  fusion  of  the  sexual 
nuclei  together  with  the  cytoplasm  of  the  gametes,  but  the  fusion  of 
the  nuclei  must  be  regarded  as  of  prime  importance. 


CHAPTER  III. 


-FECUNDATION 
ISOGAMETES. 


NON-MOTILE 


In  this  chapter  will  be  discussed  the  sexual  process  in  several  forms 
in  which  the  gametes  are  non-motile,  i.  e.,  they  do  not  escape  from 
the  parent  plant  and  move  about  in  the  surrounding  media,  and  are 
either  unisexual  or  show  a  certain  degree  of  bisexuality,  as  in  Basidio- 
bolus.  The  forms  used,  Spirogyra^  Cosmarium  and  Closterium 
among  the  desmids,  certain  diatoms  and  Basidiobolus^  have  been 
chosen  solely  because  the  development  of  the  gametes  and  their  union 
have  been  most  thoroughly  investigated  in  certain  species  of  these 
genera.  Owing  to  the  conflicting  results  obtained  by  the  several 
investigators  in  the  much-studied  Sporodinta^  the  process  in  this  plant, 
which  properly  belongs  here,  will  be  only  incidentally  referred  to. 


Fig.  19.— Copulation  of  gametes  in  Ectocarpus  siltculatus. 

A,  female  gamete  with  numerous  male  gametes  attached,  seen  from  the  side. 

B,  C,  D,  E,  successive  stages  of  cytoplasmic  fusion.— (After  Berthold.) 
E,  F,  G,  fusion  of  nucleus.— (After  Oltmanns.) 

SPIROGYRA. 

Among  the  algae  Spirogyra  undoubtedly  furnishes  the  best  known 
illustration  of  the  sexual  process  in  which  the  gametes  are  isogamous 
and  non-motile.  The  process  as  observed  in  the  living  plant  has  been 
carefully  described  long  ago  by  DeBary  ('58),  Strasburger  ('78)  and 
others,  and  it  is  now  a  matter  of  common  observation  in  almost  every 
botanical  laboratory.  The  nuclear  behavior,  which  cannot  be  fol- 
lowed in  the  living  specimen,  and  which  is  the  most  essential  part  of 
the  process,  has  received  comparatively  little  attention. 

Morphologically  and  physiologically  every  cell  of  a  Spirogyra 
filament,  except  those  serving  as  organs  of  attachment,  is  exactly  like 
every  other  cell,  so  that  the  filament  may  be  regarded,  in  a  sense  at 


68  FECUNDATION  ;     NON-MOTILE    ISOGAMETES. 

least,  as  a  colony  of  individuals.     Any  cell  of  a  filament,  save  those 
mentioned,  may  function  as  a  gamete. 

In  sexual  reproduction  cells  of  two  filaments  lying  close  side  by  side 
send  out  protuberances  toward  each  other  which  meet  end  to  end.  In 
the  contiguous  membranes  a  circular  opening  is  made  by  the  dissolu- 
tion of  the  cellulose  walls,  through  the  agency  of  an  enzyme,  whereby 
a  continuous  canal  is  formed  between  the  cells  (Fig.  20,  A).  It  is 
highly  probable  that  the  conjugating  tubes  are  brought  together  by  the 
aid, of  a  chemotactic,  directive  stimulus.  Haberlandt  ('90)  claims,  and 
his  view  is  shared  by  Klebs  ('96),  that  the  conjugating  cells  exert  a 
mutual  chemical  influence  upon  each  other,  namely,  that  a  cell  will 
put  out  a  conjugating  tube  only  when  influenced  by  another,  probably 
of  a  different  sex,  lying  near  it.  In  support  of  this  view,  Klebs  found 
that  cells  of  individual  filaments  cultivated  upon  agar-gelatin,  although 
having  been  brought  side  by  side  by  the  folding  of  the  filament,  never 
put  out  conjugating  protuberances.  A  single  male  filament,  on  the 
contrary,  may  conjugate  with  several  female  filaments  whenever  their 
cells  lie  sufficiently,  near  one  another,  but  all  those  cells  of  the  male 
filament  separated  some  distance  from  those  of  the  female  remain 
sterile  in  spite  of  the  tendency  to  conjugate.  The  limits  of  this  mutual 
action  of  the  filaments  (Haberlandt,  '90)  is  equal  to  a  distance  of  two 
or  three  diameters  of  their  cells.  Slightly  beyond  this  limit  the  cells 
may  put  out  short  conjugating  tubes,  but  these  never  reach  each  other, 
the  stimulus  being  presumably  too  weak.  Haberlandt  states  further  that 
the  conjugating  tubes  are  not  laid  down  simultaneously,  but  rather  one 
sends  out  a  protuberance  which  calls  forth  the  development  of  the  cor- 
responding tube  from  the  other  cell.  If  the  protuberances  do  not  lie 
exactly  opposite,  they  bend  slightly  in  order  to  meet  each  other.  A 
further  action  of  the  stimulus  is  seen  when  a  long  male  cell  copulates 
with  two  female  cells.  Two  canals  are  formed  connecting  the  male 
with  the  two  female  cells,  but,  of  course,  only  one  of  the  latter  receives 
the  gamete.  In  some  species,  especially  Spirogyra  inflata^  according 
to  Klebs,  the  meeting  of  the  conjugating  protuberances  is  facilitated 
by  a  curving  or  a  knee-like  bending  of  the  cells,  from  whose  convex 
sides  the  protuberances  arise. 

These  phenomena  are  not  presented  in  this  connection  for  the  purpose 
of  discussing  any  special  phase  of  the  physiology  of  the  sexual  process, 
but  merely  to  indicate  a  few  features  manifested  by  unisexual  elements 
which  show  a  tolerably  well-marked  tendency  toward  bisexuality. 

When  the  conjugation  canal,  joining  the  gametes,  is  complete,  the 
turgor  in  each  cell  is  diminished,  so  that  each  protoplast  experiences  a 
self-plasmolysis.     The  contraction  usually  takes  place  first  in  the  male 


SPIROGYRA. 


69 


gamete,  which  passes  through  the  canal  to  unite  with  the  stationary  or 
female  gamete  (Fig.  20,  A).  Strasburger  ('78)  has  observed  that 
occasionally  the  female  cell  was  the  first  to  round  up.  Haberlandt 
suggests  that  the  extrusion  of  water  is  connected  with  a  mutual 
stimulus  between  the  cells,  for  the  female  gamete  contracted  only 
when  the  male  was  normal,  and,  furthermore,  the  male  cell  became 
self-plasmolyzed  only  when  connected  with  a  female  cell. 

The  principle  underlying  the  movement  of  the  male  gamete  through 
the  canal  is  not  well  understood.  Overton  ('88)  held  that  a  gelatin- 
ous substance  was  secreted,  which,  upon  swelling,  forced  the  proto- 
plast through  the  canal.     The  presence  of  a  mucilaginous  substance 


Fig.  20. — Fusion  of  gametes  in  Spiroiyra. 

A,  portion  of  two  conjugating  filaments  of  Spiragyra  quinina. — (Aft«r 

Strasburger.) 

B,  young  zygote  provided  with  only  a  thin  wall. 

C,  zygote  at  a  later  stage ;  the  cell-wall  is  thicker,  and  the  nuclei  hav« 

united,  but  thcMiucleoli  have  not  fused. 


has  not  been  demonstrated,  however,  and  it  is  highly  probable  that  we 
have  to  do  here  with  an  active  plasmic  movement  operating  under  the 
chemotactic  stimulus  of  the  two  protoplasts.  Here  fecundation  con- 
sists in  the  union  of  the  entire  plasma  of  both  gametes,  though  DeBary 
records  the  case  of  Spiroiyra  heeriana}  in  which  a  small  vesicle  of 
plasma  is  left  beyond  the  partition  wall  in  the  conjugation  canal. 

Concerning  the  behavior  of  the  chlorophyll  bands  in  the  zygote, 
much  diversity  of  opinion  exists.  DeBary  ('58)  and  Schmitz  ('82) 
observed  that  in  species  with  one  chlorophyll  band  the  two  chloro- 
plasts  united  in  the  zygote  to  form  one  continuous  band.  Overton 
('88) ,  on  the  contrary,  asserts  that  the  single  band  of  the  female  gamete 
segments  at  the  middle  during  the  fusion  of  the  protoplasts ;  the  two 
halves  then  separate,  and  each  piece  unites  with  the  ends  of  the  band 


'  See  Fig.  lo,  p.  17,  Die  Naturlichen  Pflanzenfamilien,  i  Theil,  z  Abtheilung. 


yo  FECUNDATION  ;     NON-MOTILE    ISOGAMETES. 

furnished  by  the  male  gamete.  Chmielewskij  ('90)  finds  that  in  all  of 
the  several  species  examined  the  chloroplast  of  the  male  gamete  is 
dissolved  in  the  zygote,  that  of  the  female  only  remaining. 

The  behavior  of  the  nuclei  during  fusion  cannot  be  followed  with 
any  degree  of  certainty  in  the  living  specimen.  As  a  rule  they  cannot 
be  seen  at  all,  a  fact  which  led  to  the  view  of  the  earlier  observers  that 
the  product  of  union  was  without  a  nucleus.  One  must,  therefore, 
resort  to  thin  and  well-stained  sections  of  properly  fixed  material  to 
observe  the  details  of  nuclear  fusion.  For  this  purpose  I  have  selected 
a  small-celled  species  with  one  chlorophyll  band. 

When  the  young  zygote  is  provided  with  a  thin  cell-wall,  the  two 
nuclei,  which  are  exactly  alike,  judging  from  their  appearance,  are 
seen  lying  closely  applied  to  each  other  (Fig.  20,  B).  Each  contains 
a  rather  large  and  distinct  nucleolus  and  the  characteristic  linin  net  in 
which  are  imbedded  small  granules  that  behave  toward  stains  as 
chromatin  granules  in  resting  nuclei  of  higher  plants.  In  fact,  the 
nuclei  of  Spirogyra  in  this  condition  seem  to  possess  the  same 
structure  as  the  phanerogamic  nucleus.  The  contiguous  parts  of  the 
nuclear  membranes  dissolve  or  disappear  as  such,  and  the  network  of 
the  one  unites  directly  with  that  of  the  other,  the  fusion  of  the  nucleoli 
following  later  (Fig.  20,  C).  Frequently,  before  complete  union  of  the 
nuclei,  the  wall  of  the  zygospore  may  become  much  thickened  and  less 
easily  penetrated  by  fixing  fluids,  so  that  perfect  preparations  are  diflficult 
to  procure.  During  the  development  of  the  zygospore  the  chloroplasts 
become  vacuolate  and  the  identity  of  each  cannot  be  made  out. 

.  In  the  preceding  paragraphs  I  have  described  the  nuclear  fusion  in 
the  zygote  as  I  was  able  to  follow*5t,  but  for  lack  of  time  and  suitable 
material  an  exhaustive  study  of  the  subject  was  not  made,  and  conse- 
quently I  am  not  prepared  to  state  whether  the  peculiar  behavior  of 
the  nuclei  as  described  by  Chmielewskij  ('92)  for  Spirogyra  crassa 
and  S.  elongata  is  correct.  Chmielewskij  states  that,  as  the  gametes 
round  up,  the  nuclear  membranes  become  less  distinct,  disappearing 
entirely  as  the  gametes  unite.  The  nuclei  now  fuse,  the  fusion  being 
complete  by  the  time  the  zygote  is  provided  with  a  thick,  dark  wall. 
This  fusion  takes  place  during  the  prophase  of  division.  As  soon  as 
fusion  is  complete  the  nucleus  divides.  The  daughter  nuclei  now 
divide,  four  nuclei  resulting.  Two  of  these  then  fuse,  while  the  other 
two  divide  by  direct  division  and  finally  disorganize.  The  fusing 
nuclei  are  provided  with  membranes  and  are  in  the  resting  condition. 
If  the  observations  of  Chmielewskij  be  true,  the  process  in  Spirogyra 
is  without  parallel  in  the  plant  kingdom,  at  least  so  far  as  the  author 
is  aware. 


SPORODINIA. — CLOSTERIUM    AND    COSMARIUM.  71 

SPORODINIA. 

Morphologically  considered,  the  sexual  process  in  Sporodinta 
grandis  and  in  other  typical  Zygomycetes  seems  to  be  similar  to  that 
in  the  Conjugatece^  but  in  Sporodinia  the  gametes  are  multinucleate, 
and  the  behavior  of  the  nuclei  in  the  young  zygote  varies  considerably, 
according  to  the  accounts  given  by  the  different  obsei-vers.  After  the 
cytoplasmic  fusion  of  the  gametes,  the  nuclei  of  each  arrange  them- 
selves into  a  spherical  layer  surrounding  a  globule  of  oil,  and  then 
fuse,  producing  a  hollow  sphere  full  of  oil,  which  L6ger  ('95)  has 
called  an  embryonic  sphere  {sphere  embryonnaire) .  These  embryonic 
spheres  lie  near  the  poles  of  the  zygote.  During  the  germination  of 
the  zygospore  the  two  embryonic  spheres  fuse.  The  fused  mass 
reveals  numerous  nuclei,  which  pass  into  the  sporajigiferous  mycelium 
and  begin  to  divide.  In  the  azygospore  only  one  embryonic  sphere  is 
developed.  Wager  ('99)  regards  the  union  of  the  nuclei  to  form  the 
embryonic  sphere  as  the  sexual  act,  and  the  azygospores  are,  there- 
fore, truly  sexual,  the  process  of  conjugation  being  of  secondary 
importance.  Dangeard  ('94,  '95)  does  not  accept  Lager's  interpreta- 
tion of  the  embryonic  spheres,  holding  that  the  fate  of  the  nuclei  has 
not  been  determined. 

According  to  Gruber  ('01)  no  embryonic  spheres  are  to  be  seen  in 
the  newly  formed  zygote.  The  numerous  nuclei,  on  the  contrary,  are 
uniformly  distributed  throughout  the  cytoplasm.  After  five  or  six 
weeks  the  same  condition  of  things  was  still  found  to  exist,  and  what 
took  place  finally  among  the  nuclei  Gruber  was  unable  to  determine. 
Neither  fusion,  disorganization  nor  division  of  the  nuclei  was  observed 
even  six  months  after  the  fusion  of  the  gametes. 

From  what  is  now  known  concerning  the  sexual  union  of  multinu- 
cleate gametes  in  other  groups  of  plants,  in  which  the  sexual  process 
has  been  unmistakably  followed  in  every  detail,  it  is  very  probable  that 
a  multiple  fusion  of  the  nuclei  in  pairs  obtains  also  in  Sporodinia.^ 

CLOSTERIUM  AND  COSMARIUM. 

In  the  desmids  the  process  of  fecundation  agrees  essentially  with 
that  described  by  myself  for  Spirogyra^  except  as  regards  the  time  of 
the  fusion  of  the  sexual  nuclei  and  the  behavior  of  the  chromatophores 
in  the  zygospore.  During  the  development  of  a  firm  cell- wall  about 
the  zygote,  according  to  Klebahn  ('91),  the  chromatophores  undergo 
a  marked  change,  the  result  of  which  is  the  formation  of  two  large 
rounded  balls,  which  are  at  first  rich  in  starch  and  of  a  yellowish 
color.     The  part  taken  by  the  four  original  chromatophores  in  the 

»  Sec  Chapter  III,  Albugo  Bliti,  and  Chapter  IV,  Pyrontma. 


7a 


FECUNDATION  ;     NON-MOTILE    ISOGAMETKS. 


formation  of  these  balls  was  not  determined.  The  union  of  the  nuclei, 
which  are  in  the  resting  stage,  does  not  take  place  until  the  germina- 
tion of  the  zygote.  The  behavior  of  the  fusion  nucleus,  although 
somewhat  beyond  the  province  of  our  subject,  is  of  such  a  nature  as 


Fig.  21. — Fusion  of  sexual  nuclei  during  germination  of  the  zygote  in   Closteriutn. — (After  Klebahn.) 

A,  mature  zygote  with  two  large  chloroplasts,  the  two  sexual  nuclei  in  contact. 

B,  beginning  of  germination  ;  the  sexual  nuclei  have  fused  while  in  the  resting  condition. 

C,  contents  escaping  from  old  wall  of  zygote ;  fusion  nucleus  in  prophase  of  division. 

D,  protoplast  free  from  wall  of  zygote,  fusion  nucleus  in  anaphase  of  division. 

E,  daughter  nuclei  reconstructed,  division  of  cell  begun. 

F,  spindle  stage  of  second  mitosis  ;  the  nuclei  lie  on  opposite  sides  of  cell  near  periphery. 

G,  second  mitosis  complete. 

H,  cell-division  has  taken  place;   in  each  daughter  cell  one  of  the  two  nuclei  is  much  smaller  and 

denser;  the  two  large  nuclei  are  provided  with  membranes. 
I,  the  daughter  cells  have  begun  to  assume  form  of  adult  cell ;  in  each  the  large  nucleus  which  persists 

as  the  nucleus  of  the  cell,  has  taken  a  central  position ;  the  smaller  one  lies  near  one  end  of  cell. 

to  merit  attention,  especially  in  connection  with  the  nuclear  behavior 

previous  to  the  sexual  process  in  the  diatoms  to  be  mentioned  below. 

The  union  of  the  sexual  nuclei   in    Closterium  and  Cosmariutn^ 


CLOSTKRIUM    AND    COSMARIUM. DIATOMS. 


73 


according  to  Klebahn,  occurs  just  prior  to  the  escape  of  the  contents 
of  the  zygote  from  the  outer  membrane  (Fig.  21,  A,  B) .  During  the 
latter  process  the  fusion  nucleus  often  shows  signs  of  approaching 
karyokinesis  (Fig.  21,  C).  There  now  follow  two  karyokinetic 
divisions  in  rapid  succession,  so  that  each  daughter  cell  may  contain 
two  nuclei  (for  a  cell-division  may  also  have  taken  place)  one  of  which 
remains  as  the  nucleus  of  the  daughter  cell,  while  the  other  gradually 
undergoes  disorganization  (Fig.  21,  D,  E,  F,  G,  H,  I).  (See  expla- 
nation of  figure  for  details.) 

It  will  now  be  seen  that  the  process  in  the  zygote  of  the  desmids 
differs  from  that  described  for  Spirogyra  by  Chmielewskij  (see  p.  70) : 
(i)  in  the  fusion  of  the  sexual  nuclei  in  the  resting  stage  ;  (2)  in  that 
there  is  no  second  fusion  of  two  of  the  four  daughter  nuclei,  but  a 
cell-division,  one  nucleus  going  to  each  of  the  daughter  cells. 


/T:-^ 


B  C 

Fig.  22. — Formation  of  gametes  in  Rhopalodia  gibba. — (After  Klebahn.) 

A,  protoplast  of  cell  showing  first  mitosis  ;  nucleus  in  spindle  stage. 

B,  second  mitosis,  each  daughter  nucleus  dividing. 

C,  second  mitosis  complete,  the  four  nuclei  about  equal  in  size. 

D,  part  of  two  conjugating  individuals  ;  the  protoplast  of  the  one  on  left  has  begun  to  divide  by  becom- 

ing constricted  in  the  middle;  two  nuclei  in  each  cell  are  large,  other  two  have  become  smaller. 

E,  cell-division  complete. 

DIATOMS. 

In  the  diatoms  the  type  of  isogamous  fecundation  resulting  in  the 
formation  of  the  auxospore  recalls  the  nuclear  history  subsequent  to 
fecundation  in  the  desmids.  As  in  the  case  of  the  desmids  we  are 
indebted  also  to  the  investigations  of  Klebahn  ('96)  and  to  those  of 
Karsten  (1900),  for  a  more  accurate  knowledge  of  the  nuclear  behavior 
preceding  the  sexual  act.  The  nuclear  activity,  which  immediately 
precedes  conjugation,  is  of  prime  importance  here,  and  it  is  to  this 
that  our  attention  is  especially  directed. 

In  Rhopalodia,  the  form  studied  by  Klebahn,  two  individuals  place 
themselves  side  by  side,  being  held  together  by  means  of  mucilaginous 
masses.     The  protoplast  of  each  cell,  which  contains  one  nucleus  and 


74 


FECUNDATION  ;     NON-MOTILE    ISOGAMETES. 


in  general  two  pyrenoids,  undergoes  a  rejuvenescence  and  finally 
divides.  Prior  to  this  cell-division,  however,  two  successive  mitotic 
divisions  of  the  nucleus^  take  place  (Fig.  22,  A  to  E).  After  the  first 
mitosis  the  daughter  nuclei  generally  move  apart  toward  the  ends  of 
the  cell  whither  the  pyrenoids  also  wander  (Fig.  22,  B).  Soon  the 
second  mitosis  takes  place,  when  four  nuclei  similar  in  appearance  are 
present  in  the  protoplast,  which  may,  as  yet,  show  no  sign  of  division 
(Fig.  22,  D).  With  further  progress  the  protoplast  in  each  individual 
becomes  constricted  near  the  middle  and  finally  divides,  two  daughter 
nuclei  passing  into  each  daughter  cell,  which  contains  one  or  some- 


FiG.  23. — Conjugation  of  gametes  in  Rhopalodia  gibba. — (After  Klebahn.) 

F,  conjugating  pair  seen  from  valve  side ;  protoplast  of  each  has  divided  into  two  daughter  cells  or 

gametes  ;  each  gamete  contains,  besides  the  large  pyrenoid,  a  large  and  a  small  nucleus. 

G,  cytoplasmic  fusion  of  the  two  pairs  of  gametes  has  taken  place;  the  small  nuclei  are  scarcely  recog- 

nizable. 
H,  a  later  stage  ;  the  small  nuclei  have  entirely  disappeared,  while  the  two  functional  nuclei  in  each 
zygote,  which  has  now  changed  from  a  dumbbell  to  an  elongated  form,  have  come  nearer  together. 

times  two  pyrenoids  and  a  chromatophore  (Fig.  22,  E).  A  marked 
change  is  now  manifested  in  the  nuclei.  Of  the  two  nuclei  in  each 
daughter  cell,  one  increases  in  size  while  the  other  diminishes,  becom- 
ing dense  and  contracted  (Fig.  22,  D,  E).  The  next  step  in  the  pro- 
cess is  the  conjugation  of  the  daughter  cells  of  one  individual  with 
those  of  the  opposite  one  by  means  of  protuberances  sent  out  from 
the  respective  cells  (Fig.    23,   F,   G).     The  large  nucleus  of  each 


*  For  details  of  mitosis  see  the  original  paper  of  Klebahn,  '96. 


DIATOMS. 


75 


cell,  followed  by  the  pyrenoid,  passes  into  the  isthmus  or  connecting 
portion  of  the  dumbbell-shaped  zygote,  which  soon  becomes  cylindri- 
cal or  crescent-shaped,  and  scarcely  a  trace  of  the  small  nuclei  are 
to  be  seen  (Fig.  23,  H).  During  the  development  of  the  zygote  into 
an  auxospore,  the  two  large  functional  nuclei  assume  the  structure 
characteristic  of  the  resting  stage  (/.  e.,  each  presents  a  granular  frame- 
work and  a  definite  nucleolus)  and  fuse.  The  fusion  does  not  take 
place  in  every  case  at  a  certain  developmental  stage  of  the  two  auxo- 
spores,  but  may  occur  earlier  in  one  than  in  the  other  (Fig,  24,  I,  J). 
As  a  rule,  however,  the  fusion  is  complete  when  the  siliceous  valves 
have  begun  to  develop.  The  behavior  of  the  small  nuclei  would  seem 
to  indicate  that  they  are  utilized  as  food. 

A  slightly  different  process,  leading  to  the  production  of  the  auxo- 
spore, is  met  with  in  Cocconeis  placentula  Ehr.,  as  described  by 
Karsten  (1900).  In  this  species  the  protoplasts  of  the  conjugating 
cells  do  not  divide,  and,  therefore,  only  one  zygote  results.  In  each 
cell  there  is  also  but  one  division  of  the  nucleus  instead  of  two  as  in 
Rhopalodia.  Preparatory  to  the  cytoplasmic  union  the  protoplast  of 
each  cell  contracts.  Each  cell  is  seen  to  possess  two  nuclei,  one  large 
and  one  small,  so  that  nuclear  division  must  have  taken  place  at  an 
earlier  stage.  During  the  contraction  mentioned  each  protoplast  sur- 
rounds itself  with  a  gelatinous  envelope.  Near  the  point  of  contact  of 
the  two  individuals  the  two  halves  of  each  shell  separate  slightly.  From 
the  opening  in  one  of  the  cells,  which  is  regarded  as  the  male  gamete, 
a  small  papilla  protrudes,  which  grows  toward  the  opening  in  the 
female  cell,  and  the  gelatinous  envelopes  are  soon  in  open  communi- 
cation. The  entire  protoplast  of  the  male  cell  now  passes  through 
this  narrow  channel  into  the  female  cell.  The  young  zygote  then 
increases  considerably  in  size,  and  begins  the  formation  of  a  firm  cell- 
wall  about  itself.  Of  the  four  nuclei  only  the  two  large  ones  are  now 
to  be  seen,  the  smaller  ones  having  gradually  disappeared.  The  two 
large  functional  nuclei,  each  with  a  nucleolus,  begin  to  fuse  slowly, 
and,  by  the  time  the  shell  of  the  zygote  is  fully  formed  and  the  two 
chromatophores  are  reduced  to  one,  fusion  is  complete. 

From  the  foregoing  it  is  clear  that  the  nuclear  behavior  immediately 
preceding  the  sexual  act  in  Rhopalodia  is  strikingly  analogous  to 
the  process  following  fecundation  in  Closterium  and  Cosmarium. 
Whether  these  processes  bear  any  closer  relation  to  each  other  than 
mere  analogy  is  a  difficult  question.  It  may  be  suggested  that,  in  the 
case  of  the  diatoms,  we  have  to  do  with  the  development  of  two  perfect 
gametes  in  each  cell  instead  of  four,  a  process  similar  to  that  in  certain 
Fucacece^  where  only  part  of  the  egg-cells  in  the  oogonium  mature, 


76 


FECUNDATION  ;     NON-MOTILE    ISOGAMETES. 


the  others  being  disorganized ;  and  in  the  desmids  only  two  out  of  the 
four  in  the  germination  of  the  zygote  develop  into  perfect  cells. 

It  is  not  known  whether  the  reduction  in  the  number  of  chromo- 
somes, if  a  reduction  actually  occurs  in  either  desmids  or  diatoms,  is 
in  any  way  associated  with  the  nuclear  divisions  in  question,  as  has 
been  assumed  by  some  authors  (see  Wilson,  "The  Cell,"  p.  198)  ; 
consequently,  in  the  light  of  our  present  knowledge,  it  cannot  be  said 
with  any  certainty  that  these  nuclear  divisions  represent  a  preparation 
for  the  sexual  act,  that  in  the  diatoms  taking  place  just  before  fecun- 
dation while  in  the  desmids  it  occurs  at  the  beginning  of  an  ontoge- 
netic development. 

BASIDIOBOLUS. 

A  sexual  process  similar  to  that  in  the  Conjugatecs  is  found  in 
Basidiobolus,  one  of  the  Phycomycetes.     I  have  selected  Basidio- 


Fio.  34.— Fusion  of  the  sexual  nuclei  in  Rk0f»~ 
lodia £ih6a.—{A.f\.e.r  Klebahn.) 
I,  the  two  young  zygotes  or  auxospores  have  elon- 
gated  and  begun  to  assume  form  of  adult;  sexual 
nuclei  now  in  contact. 
J,   middle  portion  of  two  auxospores,  each  with   a 
fusion  nucleus. 

bolus  ranarum  because  of  its  close  re- 
semblance to  certain  Mesocarfacece^ 
especially  Mougeotia^  both  in  structure 
(the  cells  possess  only  one  nucleus)  and 
in  the  sexual  process,  and  because  the  development  of  the  sexual 
organs  and  the  fusion  of  the  gametes  are  well  known  in  detail.  Sex- 
uality in  this  genus  has  recently  been  subjected  to  a  critical  study  by 
Fairchild  ('97)?  whose  results  form  the  basis  of  the  following  account. 
Two  neighboring  cells  of  a  filament  send  out  near  the  transverse 
wall  a  beak-like  protuberance,  into  which  the  nuclei  of  the  respective 
cells  pass  (Fig.  25,  A). 

The  nucleus  in  each  of  the  protuberances  now  undergoes  a  karyo- 
kinetic  division,  which  is  followed  by  the  formation  of  a  transverse 


BASIDIOBOLUS. 


11 


wall  cutting  off  a  small  cell  at  the  end  of  the  beak  (Fig.  25,  B).  The 
manner  in  which  this  wall  is  laid  down  is  worthy  of  special  notice 
here,  since  it  is  formed  as  in  the  higher  plants,  namely,  through  the 
instrumentality  of  the  kinoplasmic  connecting  fibers,  appearing  at 
first  as  a  cell-plate.  Apart  from  Chara  this  is  the  only  instance 
as  yet  known  among  the  lower  cryptogams  in  which  a  cell-plate  is 
thus  formed.  Immediately  the  nuclei  have  entered  the  beaks,  and 
prior  to  the  prophase  of  the  nuclear  division  just  mentioned,  and  also 
before  an  increase  in  size  of  the  female  gamete,  a  hole  is  formed  in 
the  transverse  wall  separating  the  two  gametes. 

The  two  daughter  nuclei  cut  off  in  the  ends  of  the  beaks  gradually 
disappear,  while  the  other  two  pass  down  deeper  into  the  cytoplasm 


Fig.  25.— Formation  of  gametes  in  Basidiobolut  ratutrum  Eidam.— {After  Fairchiid.) 

A,  two  gametes  showing  the  beaks;  the  nuclei,  which  are  in  the  beaks,  are  in  the  resting  condition  ; 

the  hole  has  already  formed  between  the  gametes. 

B,  the  nuclei  have  divided  and  two  of  the  daughter  nuclei  are  cut  off  in  the  ends  of  the  beaks,  while  the 

other  two,  which  have  increased  in  sire,  have  passed  down  near  the  opening  in  the  transverse  wall  ; 
the  female  gamete  has  increased  greatly  in  size,  the  male  retaining  its  former  dimensions. 

of  the  cells  (Fig.  25,  B).  The  male  nucleus  now  passes  through  this 
opening  and  comes  in  contact  with  the  female  nucleus  (Fig.  26,  C) . 
During  these  movements  the  nuclei  attain  their  original  size,  and  each 
contains  one  or  more  interwoven  nuclear  threads,  in  which  chromatin 
granules  are  situated  at  rather  long  intervals.  In  this  condition  the 
two  nuclei  remain  some  time  before  fusing.  The  entire  cytoplasm 
of  the  two  gametes  is  utilized  in  the  formation  of  the  young  zygospore, 
which  now  forms  about  itself  a  very  thin  wall,  within  which  the 
thick  endospore,  consisting  of  several  layers,  is  gradually  developed. 
Owing  to  the  difficulty  with  which  fixing  fluids  penetrate  the  thick 
wall  of  the  zygote  the  exact  time  of  fusion  of  the  male  and  female 
nuclei  is  not  easily  determined,  but  as  the  zygospore  approaches 
maturity  the  fusion  is  complete,  so  that  no  trace  of  male  and  female 


78 


FECUNDATION  ;     NON-MOTILE    ISOGAMETES. 


nuclei  can  be  distinguished  (Fig.  26,  D).  According  to  Raciborski 
('96)  the  fusion  may  be  delayed  until  the  germination  of  the  zygote. 
The  full  significance  of  the  formation  of  the  beaks  into  which  the 
nuclei  wander,  the  division  of  the  latter,  and  the  cutting  off  of  the 
small  cells  which  degenerate,  can  be  more  fully  understood  only  after 
the  process  of  sexual  reproduction  is  known  in  other  and  related  forms. 
The  two  small  cells  cut  off  in  the  ends  of  the  beaks  may,  however,  be 


Fig.  26. — Fusion  of  sexual  nuclei  in  Basidiobolus  ranarum. — (After  Fairchild.) 
C,  the  sexual  nuclei  are  in  contact.         D,  zygote  with  fusion  nucleus  and  thick  cell-wall. 

reasonably  regarded  as  degenerate  gametes,  although  it  may  seem  idle 
to  attempt  to  explain  or  to  bring  into  line  the  various  peculiar  phenom- 
ena brought  out  in  the  several  preceding  paragraphs  that  pertain  to 
the  desmids,  diatoms,  Basidiobolus  and  Spirogyra.  In  the  desmids, 
diatoms  and  Basidiobolus^  it  is  possible  that  all  these  phenomena 
may  have  resulted  independently  from  similar  causes  acting  during  a 
large  part  of  the  phylogenetic  history  of  the  respective  groups  of  plants. 


CHAPTER  IV.— FECUNDATION  ;  HETEROGAMETES. 

In  the  preceding  chapters  we  have  considered  sexual  reproduction 
in  certain  of  those  Thallophyta  in  which  no  very  marked  differentia- 
tion of  the  gametes  has  been  attained,  although  in  Bctocarpus  espe- 
cially, and  even  in  Sfirogyra  and  Bastdiobolus,  a  tendency  toward 
a  differentiation  into  male  and  female  cells  is  manifested.  Nor  have 
we  found  any  modification  of  the  cells  bearing  the  gametes  into  dif- 
ferentiated sexual  organs,  unless  the  gametangia  of  such  forms  as 
Ectocarpus  be  so  considered,  and  even  then  there  is  no  apparent 
difference  between  male  and  female  gametangia.  As  already  men- 
tioned in  the  introductory  chapter,  the  terms  male  zx^^  female  sexual 
cells  are  essentially  the  expression  of  a  certain  fundamental  kind  of 
division  of  labor,  and  in  the  developmental  history  of  sexuality  in 
plants  we  find  this  division  of  labor  manifested  in  the  gametes  them- 
selves before  a  corresponding  differentiation  is  apparent  in  the  organs 
bearing  them. 

SPH^ROPLBA. 

Among  the  algae  one  of  the  best  known  and  most  interesting  exam- 
ples of  this  fact  is  illustrated  in  Sphceroflea  annulina.  To  Ferdinand 
Cohn  ('55)  is  due  the  credit  of  having  established  the  fact  of  sexual 
reproduction  in  this  genus,  a  phenomenon  among  the  algae  little  known 
at  the  time.  Later  Sphceroplea  was  studied  by  Heinricher  ('83), 
Rauwenhoff  ('88),  Kny  ('84)  and  more  recently  by  Klebahn  ('99). 
Although  both  Heinricher  and  Rauwenhoff  followed  the  behavior  of  the 
nucleus  during  certain  stages  in  the  development  of  the  sexual  cells  and 
in  fecundation,  yet  in  many  respects  their  work  was  incomplete.  For 
a  more  thorough  investigation  of  this  process,  however,  we  are  indebted 
to  the  researches  of  Klebahn,  who  studied  the  two  varieties  of  the 
species,  S.  annulina  var.  braunii  (Keutz)  Kirchner  and  S.  annulina 
var.  crassisepta  Heinricher.  The  chief  interest  in  the  sexual  repro- 
duction of  this  plant  centers  upon  the  fact  that  in  var.  braunii  several 
nuclei  are  usually  present  in  the  egg-cell. 

The  contents  of  the  multinucleate  cells  of  Sphcsroplea  present  the 
well-known  and  characteristic  arrangement :  In  typical  cases  the  cen- 
tral cavity  of  each  cell  is  traversed  by  a  row  of  large  vacuoles  inter- 
spersed by  smaller  ones  of  varying  size.  The  protoplasm,  which  forms 
only  a  thin  layer  between  the  larger  vacuoles  and  the  cell-wall,  is 
collected  into  dense   ring-like   or  band-shaped    masses  between   the 


So 


FECUNDATION ;  HETEROGAMETES. 


former.  These  plasmic  rings  or  diaphragms  communicate  with  each 
other  by  plasmic  strands  or  bridges.  In  the  plasmic  rings  are  located 
the  rounded  chloroplasts,  pyrenoids  and  the  nuclei.  Of  the  latter  the 
number  in  each  ring  varies  from  3  to  20  in  var.  draum'i  and  from  i  to  4 
in  var.  crassisepta  (Fig.  27,  A). 

In  those  cells  in  which  spermatozoids  are  developed  the  nuclei 
undergo  four  or  five  karyokinetic  divisions,^  so  that  ultimately  about 
300  small  nuclei  are  present  in  each  band  (Fig.  28,  A  to  F).  During 
these  divisions  the  pyrenoids  disappear,  and  the  chromatophores 
undergo  several  divisions  and  assume  a  pale,  yellowish-brown  color. 


Fig.  87. — Cell-cleavage  leading  to  formation  of  egg-cells  in  SpheeropUa  braunii. — (After  Klebahn.) 

A,  outer  view  of  a  protoplasmic  ring  of  a  vegetative  cell, showing  chromatophores,  pyrenoids  and  nuclei. 

B,  portion  of  an  odgonium  showing  frothy  nature  of  protoplasm  and  early  stages  of  cleavage. 

C,  small  portion  of  oogonium,  showing  irregular  protoplasts  resulting  from  cleavage,  which  contain 

several  nuclei  and  pyrenoids. 


The  plasmic  rings  up  to  this  time  retain  their  original  form.  Now 
the  cytoplasm  segments  into  numerous  protoplasts,  the  spermatozoids, 
in  such  a  manner  that  each  spermatozoid  receives  only  one  nucleus 
(Fig.  29,  I,  J,  K,  L).  The  mature  spermatozoids  (var.  crassisepta) 
are  as  a  rule  spindle-shaped,  being  smaller  at  the  anterior  end,  which 
bears  the  two  cilia.  Near  the  middle  lies  the  veiy  small  and  densely 
staining  nucleus  (Fig.  29,  L).  Kny  in  his  Wandtafel,  Lxiir,  figui-es 
four  or  five  yellowish  chromatophores  in  each  spermatozoid. 

The  processes  leading  to  the  formation  of  the  egg-cells  show  a 
marked  difference  from  those  taking  place  in  the  antheridium.     Even 


'  For  details  of  karyokinesis  see  Klebahn,  '99. 


SPH^ROPLEA. 


8l 


in  the  two  varieties,  as, will  be  shown,  the  cleavage  is  not  the  same. 
In  var.  braunii  the  ring-like  disposition  of  the  protoplasm  disappears, 
while  large  vacuoles  appear,  transforming  the  entire  cell-contents  into 
a  foamy  structure  in  which  larger  and  smaller  strands  and  masses 
alternate  (Fig.  27,  B).  In  the  dense  portions  of  protoplasm  nuclei,  as 
well  as  chromatophores  and  pyrenoids,  are  irregularly  disposed.  Now 
a  cleavage  takes  place  by  which  the  plasmic  contents  are  segmented 
into  irregular  protoplasts  of  varying  sizes  (Fig.  27,  C).  These  proto- 
plasts contract  (the  large  vacuoles  thereby  gradually  disappearing)  and 


Fig.  a8. — Parts  of  contents  of  young  antheridia,  showing 
nuclear  history  preparatory  to  formation  of  sperma- 
tozoids  in  S.  braunii. — (After  Klebahn.) 

A,  part  of  plasmic  ring  showing  two  nuclei  in  prophase  of 
division. 

B,  spindle  stage  of  same  mitosis. 

C,  anaphase  probably  from  second  mitosis. 

D,  Telophase  of  a  later  nuclear  division. 

E,  Condition  of  nuclei  between  successive  mitoses,  pyre- 
noids still  present. 

F,  nuclei  shortly  before  formation  of  spermatozoids ;  the 
pyrenoids  have  disappeared. 


round  up  to  form  the  egg-cells,  of  which  two  to  four  are  seen  in  a  cross- 
section  of  the  cell. 

Neither  shortly  before  nor  during  cleavage,  according  to  Klebahn 
('99),  is  there  to  be  observed  a  division  or  fusion  of  the  nuclei,  so  that 
(contrary  to  Rauwenhoff  who  claimed  that  during  the  formation  of  the 
eggs  the  number  of  nuclei  was  diminished)  each  egg'  may  contain,  in 
addition  to  2  or  more  pyrenoids,  several  nuclei,  the  number  varying 
from  I  to  5  (Fig.  29,  A  to  E) .  The  number  of  nuclei  falling  to  any 
egg  is  largely  a  matter  of  chance,  since  the  cleavage  planes  do  not  seem 
to  be  determined  in  any  way  by  the  number  or  position  of  the  nuclei 
in  the  cytoplasm. 


*  The  so-called  "giant  eggs"  are  exceptions. 


82  FECUNDATION  ;  HETEROGAMETES. 

In  var.  crasstsepta^  whose  cells  are  smaller  (narrower)  and  with 
fewer  nuclei,  the  process  of  cleavage  differs  somewhat.  The  eggs  in 
this  variety  contain,  as  a  rule,  only  one  nucleus.  When  the  protoplasm 
of  the  oogonium  has  become  frothy,  as  described  for  var.  braunii^ 
cleavage  planes  are  formed  at  right  angles  to  the  long  axis  of  the  cell, 
thus  separating  the  contents  into  a  row  of  short  segments.'  Here  the 
cleavage  follows  in  such  a  way  that  a  nucleus  will  be  included  in  each  seg- 
ment of  the  cell,  although  in  exceptional  cases  two  nuclei  may  be  included 
in  a  segment.  In  var.  braunii  we  have,  therefore,  to  do  with  multinu- 
cleated eggs,  while  in  var.  crassisepta  each  egg-cell  is  uninucleate. 

When  the  egg-cells  are  mature,  small  openings  are  formed  in  the 
wall  of  the  oogonium  through  which  numerous  spermatozoids  enter 
(Kny,  Wandtafel,  lxiv).  The  manner  in  which  the  spermatozoids 
unite  with  the  cytoplasm  of  the  egg  was  not  observed  by  the  authors 
cited.  According  to  Klebahn  ('99)  the  fecundated  egg  is  readily  dis- 
tinguished by  its  delicate  membrane  and  by  the  presence  of  the  sperm 
nucleus  which  appears  always  in  sharp  contrast  to  the  nuclei  of  the  egg 
(these  resemble  vegetative  nuclei)  as  a  small,  densely  staining  body 
about  the  size  of  the  nucleolus  (/.  ^.,  about  one  micron  in  diameter) 
(Fig.  29,  A,  B).  In  eggs  just  fecundated  the  sperm  nucleus  lies  at  the 
surface  beneath  the  delicate  membrane.  After  a  time,  the  length  of 
which  was  not  determined,  the  sperm  nucleus  passes  into  the  interior 
of  the  egg,  and  finally  fuses  with  one  of  its  nuclei  (Fig.  29,  C,  D,  E). 
Before  actual  fusion  the  two  sexual  nuclei  remain  side  by  side  some 
time,  a  phenomenon  of  very  frequent  occurrence  in  the  plant  kingdom, 
during  which  the  male  nucleus  increases  in  volume,  its  chromatic  sub- 
stance assuming  the  form  of  larger  and  more  distinct  granules,  until 
finally  the  two  sexual  nuclei  can  scarcely  be  distinguished  one  from  the 
other.  The  fusion  nucleus  is  easily  recognized  by  its  coarsely  granular 
contents,  while  the  other  nuclei  in  the  egg  appear  pale,  with  a  few  small 
granules  arranged  along  the  nuclear  membrane  (Fig.  29,  F). 

From  the  foregoing  it  will  be  seen  that  in  Sph(^roplea  annulina  var. 
braunii^  although  several  nuclei  are  present  in  the  ^%%^  fecundation 
consists  in  the  fusion  of  the  spermatozoid  nucleus  with  only  one  nucleus 
of  the  egg-cell.  Whether  there  exists  among  the  several  nuclei  of  the 
egg  any  preference  in  the  union  with  the  male  nucleus  is  not  known,  as 
there  seems  to  be  nothing  in  the  position  or  appearance  of  the  nuclei 
which  might  suggest  a  preference.  The  nuclei  are  irregularly  grouped 
or  distributed  in  the  cytoplasm  of  the  egg,  and  it  seems  to  be  purely  a 
matter  of  chance  as  to  which  one  will  fuse  with  the  sperm  nucleus. 


See  Kny'i  Wandtafel,  lxiy. 


SPH^ROPLEA. 


83 


After  fusion  of  the  sexual  nuclei  the  oospore  develops  its  character- 
istic wall  (Fig.  29,  G,  H).  Unfortunately  Klebahn  was  unable  to 
trace  the  fate  of  the  remaining  nuclei.  Whether  they  disappear  indi- 
vidually or,  after  fusion  with  each  other,  unite  with  the  fusion  nucleus, 
is  a  matter  of  conjecture  only.     The  investigations  of  Golenken  (1900) 


Fig.  29.— Fecundation  of  eggs  and  later  development  of  spermatoroids.    A-H,  SpkaropUa  braunii. 
I-M,  S.  crasshe/ita.—I^MtKT  Klebahn.) 

A,  egg  with  3  nuclei,  into  which  a  sperm  has  just  penetrated. 

B,  same  stage  as  A ;  egg  with  5  nuclei, 

C,  egg  with  4  nuclei  and  5  pyrenoids ;  the  sperm  nucleus  has  penetrated  farther  into  egg 

D,  sperm  nucleus  applied  to  functional  nucleus  of  egg. 

E,  fusion  of  two  sexual  nuclei. 
F-H,  maturation  of  oospore. 

I-K,  later  stages  in  development  of  spermatozoids. 

L,  two  spermatozoids. 

M,  part  of  an  oogonium  showing  fecundated  eggs  and  spermatozoids  within. 

seem  to  throw  further  light  upon  the  subject.  As  reported  in  the 
Botanisches  Centralblatt,  84,  p.  284,  1900,  this  author,  who  observed 
the  sexual  process  in  a  variety  of  Sphceroflea  annulina,  which  con- 
tained multinucleate  as  well  as  uninucleate  eggs,  finds  that  m  the 
multinucleate  eggs  the  nuclei  lie  near  each  other  close  to  the  surface, 
and  at  a  spot  where  the  spermatozoids  seem  to  enter.  After  fecunda- 
tion the  nuclei  first  distribute  themselves  regularly  within  the  egg  and 
then  finally  fuse  to  form  one  nucleus. 


84  FECUNDATION  ;  HETEROGAMETES. 

In  var.  crassisepta  with  uninucleate  egg-cells  the  problem  is  simpler. 
The  observation  of  the  process  in  this  form  in  connection  with  var. 
braunii  was  fortunate,  as  it  must  have  served  as  a  control  in  the 
interpretation  of  the  phenomena  in  the  multinucleate  eggs.  If  the 
observations  of  Klebahn  be  correct,  var.  braunii  represents  the  only 
authentic  case  among  the  algas  of  a  normal  sexual  union  of  a  single 
male  and  female  nucleus  in  an  egg-cell  containing  several  nuclei  of 
apparently  equal  morphological  value. 

FUCACEiE. 

In  certain  respects  the  sexual  process  in  Sphceroplea  is  suggestive 
of  that  in  the  Fucacece.  In  the  latter,  however,  we  have  the  addi- 
tional feature  that  the  female  gametes  or  eggs  escape  into  the  water, 
and  copulation  takes  place  outside  of  the  oogonium.  Probably  no 
other  representative  of  the  algae  is  so  favorable  for  the  observation 
of  the  external  phenomena  of  the  sexual  process  than  is  Fucus. 

The  more  obvious  details  of  the  process  have  been  observed  by 
Thuret,  Oltmanns  and  others,  but  it  is  to  the  recent  researches  of  Far- 
mer and  Williams  ('96,  '98)  that  we  are  indebted  for  a  thorough 
and  comprehensive  account  of  the  phenomena  to  be  observed  in  the 
living  material.  The  work  of  these  authors  supplements  also  the 
observations  of  Strasburger  ('97)  on  the  development  of  the  gametes 
and  on  the  behavior  of  the  sperm-nucleus  after  it  enters  the  egg. 

The  type  of  division  of  the  cell  and  nucleus  in  the  development  of 
the  gametes  in  this  group  of  plants  has  been  fully  treated  in  the  intro- 
ductory chapter,  and  the  escape  of  the  egg-cells  from  the  oogonium  is 
too  well  known  to  bear  repetition  in  this  place. ^  Since,  however, 
Fucus  has  figured  prominently  in  recent  and  much  discussed  theories 
bearing  upon  the  significance  of  the  number  of  the  chromosomes  in 
sex  and  heredity,  it  is  probably  not  out  of  place  here  to  state  that,  in 
the  first  nuclear  division  in  the  oogonium,  the  reduced  number  of 
chromosomes  appears,  and  that  both  the  nucleus  of  the  egg  and  the 
spermatozoid  contain  this  number. 

In  order  to  observe  the  behavior  of  the  sexual  cells  while  alive,  and 
to  obtain  suitable  material  for  the  indirect  method  of  study.  Farmer 
and  Williams  state: 

Male  and  female  plants  were  kept  in  separate  dishes,  and  were  covered  to 
prevent  drying  up.  ...  On  the  appearance  of  the  extruded  products,  the 
female  receptacles  were  placed  in  sea-water,  and  after  the  complete  liberation 
ot  the  oospheres  a  few  male  branches  with  ripe  antherozoids  were  first  placed 

'  On  the  method  of  the  liberation  of  the  sexual  cells,  see  Farmer  and  Williams,  '98,  p.  629. 


FUCACE^.  85 

in  a  capsule  of  seawater  until  it  became  turbid  owing  to  their  number.  If  on 
examination  the  antherozoids  proved  to  be  active,  small  quantities  were  added 
to  the  vessel  containing  the  oospheres.     ('96,  p.  480.) 

When  vigorous  antherozoids  (1.  c,  '98,  p.  631)  are  transferred  to  vessels  con- 
taining healthy  oospheres  they  at  once  congregate  around  them,  and  attaching 
themselves  to  the  periphery  of  the  eggs,  cause  the  well-known  movements  by 
lashing  the  water  with  the  free  cilium.  But,  as  Thuret  noticed,  fertilization  can 
often  be  effected  without  any  whirHng  movement  taking  place,  and  we  have 
observed  perfectly  normal  germination  to  follow  on  the  addition  of  apparently 
inactive  antherozoids  to  the  oOspheres. 

There  seems  to  be  a  marked  difference  between  the  degree  of  attrac- 
tion exerted  on  the  antherozoids  by  the  egg-cells  under  different  condi- 
tions. Thus,  when  the  extruded  products  have  been  long  exposed  to 
a  moist  atmosphere,  so  that  all  the  membranes  have  become  deli- 
quescent, the  spermatozoids  are  hardly  influenced  by  the  oospheres. 
On  the  other  hand  the  oospheres  which  still  retain  their  walls  become 
covered  with  spermatozoids. 

The  behavior  of  the  spermatozoids  in  the  genus  Halidrys  is  of 
especial  interest  in  this  connection,  and  I  quote  again  from  the  same 
authors  (1.  c,  '98,  p.  633) : 

On  watching  the  behavior  of  the  antherozoids  when  swimming  amongst  the 
oospheres,  they  are  seen  to  attach  themselves  to  the  surface  of  the  eggs  by  one 
cilium,  whilst  they  maintain  a  circular  or  gyratory  movement  around  their 
point  of  attachment.  Most  often  there  is  a  number — a  dozen  or  more — of 
these  groups,  each  consisting  of  4  to  12  antherozoids,  distributed  over  the  sur- 
face of  each  odsphere.  The  movement  is  always  in  the  clockwise  direction, 
and  the  chromatophore  is  on  the  end  of  the  antherozoid  remote  from  the  egg. 
The  rate  of  gyration  is  fairly  rapid,  40  to  50  complete  turns  being  made  in  a 
minute.  After  this  has  been  going  on  for  a  while  the  egg  itself  evinces 
a  change,  swelling  somewhat  and  appearing  more  transparent  than  before. 
Sometimes  movements  of  vacuoles  may  be  discerned,  and  even  the  position  of 
the  nucleus  may  change.  These  alterations  ensue  as  the  definite  result  of  the 
stimulus  in  some  way  given  by  the  antherozoids  themselves.  .  .  .  Sud- 
denly the  antherozoids  are  seen  to  leave  the  &gg  like  a  crowd  of  startled  birds, 
or  else  they  become  quiescent,  and  these  phenomena  are  immediately  followed 
by  a  great  change  in  the  egg  itself,  which  becomes  warty  and  covered  with 
conical  projections.  From  each  papilla  a  fine  thread  projects,  consisting  of  a 
moniliform  series  of  droplets,  and  the  antherozoids  may  sometimes  be  observed 
attached  to  these  threads.  After  the  lapse  of  a  few  (3  to  5)  minutes  the  egg 
resumes  its  spherical  form  whilst  at  the  same  time  its  diameter  becomes 
smaller.  Still  later  the  fine  threads  also  disappear,  whilst  the  egg  regains  its 
original  size.  As  long  as  the  antherozoids  are  in  active  motion  on  the  surface 
of  the  egg,  the  latter  exhibits  a  scarcely  perceptible  rocking  movement  and  is 
free  in  the  water,  but  during  the  events  which  have  just  been  narrated  it 


86  FECUNDATION  ;  HKTEROGAMETES. 

becomes  attached  to  the  surface  on  which  it  may  be  resting.  We  consider  it 
as  certain  that  the  flight  of  the  supernumerary  antherozoids  marks  the  moment 
of  actual  fertilization,  and  it  seems  only  possible  to  interpret  the  events  outside 
the  egg  as  the  results  of  an  excretion  from  it  of  some  substance  which  not  only 
exerts  on  the  surrounding  antherozoids  a  negative  chemotactic  but  also  a 
directly  injurious  effect,  for  a  number  of  dead  sperms  may  be  seen  around  the 
fertilized  egg.  Possibly  the  bead-like  filaments  which  partly  stain  like  muci- 
lage, are  directly  concerned  in  the  process. 

The  facts  observed  by  Farmer  and  Williams  have  been  given  some- 
what in  detail,  because  they  are  suggestive  of  various  interesting 
problems,  especially  those  pertaining  to  chemotaxis  between  sexual 
cells,  a  province  of  physiology  well  worthy  of  careful  investigation, 
and  one  which  will  undoubtedly  yield  fruitful  results. 

It  may  be  noted  that,  in  the  attachment  of  the  spermatozoids  to  the 
egg  by  means  of  one  cilium,  and  in  the  sudden  withdrawal  of  the  super- 
numerary sperms  as  if  startled,  a  certain  resemblance  exists  between 
Halidrys  and  Ectocarpus  (see  p.  ^d),^  although  these  phenomena 
are  less  marked  in  the  latter. 

In  the  case  of  normal  healthy  products,  fecundation  occurs  within  a 
few  minutes  after  the  addition  of  the  male  cells.  The  fecundated  eggs 
form  a  membrane  around  themselves  at  once,  and  behave  in  a  very 
different  manner  from  those  into  which  no  spermatozoids  have  pene- 
trated. For  example,  if  the  sea-water  be  gradually  drawn  off  from  a 
mixture  of  fecundated  and  non-fecundated  eggs,  the  latter  flatten  out, 
their  cytoplasm  loses  its  coherence  and  becomes  distributed  in  all 
directions,  while  the  former  show  only  local  protuberances  and  burst 
only  at  one  point. 

The  passage  of  the  sperm-nucleus  through  the  cytoplasm  and  its 
fusion  with  the  nucleus  of  the  egg  can  be  followed  with  anything  like 
accuracy  only  in  thin  and  properly  stained  sections.  According  to 
Strasburger  ('97),  the  egg  of  JFucus  platycarpus  at  the  time  of  fecun- 
dation is  globular  and  p^rovided  with  only  a  plasma  membrane.  The 
alveoli  of  its  cytoplasm,  together  with  the  included  chromatophores, 
are  radially  disposed  about  the  centrally  placed  nucleus  (Fig.  30,  A), 
an  arrangement  which  seems  to  facilitate  the  movement  of  the  sperm 
to  the  egg-nucleus.  The  passage  of  the  sperm  through  the  cytoplasm 
and  its  union  with  the  nucleus  of  the  egg  take  place  rapidly,  for  both 
Strasburger  and  Farmer  agree  that  ten  minutes  after  the  addition  of 
the  spermatozoids  to  the  water  containing  the  eggs  the  sexual  nuclei 
have  united.  Strasburger  is  inclined  to  the  view  that  th?  larger  por- 
tion of  the  cytoplasm  of  the  spermatozoid  on  entering  the  Q^<g  unites 


FUCACK^ . 


87 


with  its  cytoplasm,  while  the  nucleus  alone  proceeds  toward  that  of 
the  egg.  However,  the  body  which  approaches  the  egg-nucleus  is 
wedge-shaped  or  narrowed  slightly  at  one  end.  When  the  sperm- 
nucleus  reaches  that  of  the  egg  it  is  about  the  size  of  the  nucleolus  of 
the  latter  (Fig.  30,  A).  It  appears  as  a  densely  stained  and  somewhat 
flattened  or  lens-shaped  body  closely  applied  to  the  egg-nucleus  (Fig. 
30,  B).  An  increase  in  size  now  follows,  during  which  the  denser 
appearance  gives  way  to  that  of  a  less  compact  structure  (Fig.  30,  C) . 
It  is  now  seen  (Strasburger,  '97,  p.  364)  that  the  sperm-nucleus 
possesses  a  thread-like  framework.     With  further  increase  in  size  the 


D  ^-^^^  c 

Fig.  30.— Fecundation  in  Fucus.     A-D,  Fucus  vesiculosus.     E,  F.  serratus —lAhcr  Strasburger.) 

A,  Egg-cell  ten  minutes  after  mixing  of  sexual  elements  ;  male  nucleus  applied  to  that  of  egg. 

B,  same  two  nuclei  more  highly  magnified. 

C,  similar  to  B;  sperm  nucleus  lies  between  the  observer  and  egg-nucleus. 

D,  fusion  of  nuclei  has  progressed  further ;  lo  minutes  after  mixing  of  sexual  elements. 

E,  1  J$  days  after  fecundation ;  fusion  nucleus  preparing  for  division  ;  poles  of  future  spindle  present, 

but  limits  of  the  two  nuclei  still  recognizable ;  the  part  derived  from  male  nucleus  (on  the  left)  has  also 
a  nucleolus. 

chromatin  thread  becomes  more  prominent,  and  the  boundary  between 
sperm  and  egg-nucleus  gradually  disappears  (Fig.  30,  D,  E).  In  the 
meantime  a  nucleolus  is  found  in  that  portion  of  the  fusion-nucleus 
coming  from  the  sperm.  This  is  in  all  probability  not  brought  in 
as  such,  but  is  developed  during  the  process  of  fusion  much  in  the 
same  way  as  in  the  reconstruction  of  daughter-nuclei  following  karyo- 

kinesis. 

In  no  case  observed  by  the  authors  mentioned  was  the  sperm-nucleus 
accompanied  by  a  centrosphere  or  a  system  of  radiations,  either  during 
its  passage  through  the  cytoplasm  or  during  fusion.  Strasburger  ('97, 
p.  365)  states,  however,  that  in  some  cases  he  was  able  to  truce  the 
apparent  connection  between  the  two  centrospheres  and  the  limits  of 
the  two  sexual  nuclei  in  the  oospore  (Fig.  30,  E),  and  he  infers  that 
the  centrosomes  may  have  been  brought  into  the  egg  by  the  sperm  in 


S8  FECUNDATION  ;  HETEROGAMETES. 

an  unrecognizable  condition.  In  the  light  of  what  is  known  in  certain 
animal  eggs  such  an  inference  was  tempting,  but,  from  our  present 
knowledge  of  the  centrosphere  and  centrosome  in  plants,  such  a  con- 
clusion is  no  longer  justifiable.  Moreover,  when  the  centrospheres 
appear  in  the  first  nuclear  division  of  the  fecundated  egg,  it  is  difficult, 
and  may  be  impracticable,  to  distinguish  between  the  male  and  female 
portions. 

Only  in  rare  cases  does  more  than  one  spermatozoid  enter  the  egg, 
for  among  several  thousand  preparations  examined  by  Farmer  and 
Williams,  only  three  cases  of  polyspermy  were  observed  in  which  two 
spermatozoids  had  effected  an  entrance.  The  rare  occurrence  of  poly- 
spermy under  such  conditions  as  are  normal  for  the  plants  concerned, 
and  as  appears  favorable  for  this  phenomenon,  would  seem  to  indicate 
that  many  cases  of  polyspermy  reported  for  animals  might  be  largely 
the  result  of  the  prevalence  of  abnormal  conditions  at  the  time  of 
fecundation. 

Concerning  the  large  oosphere-like  bodies  with  two  nuclei  in  Fucus^ 
which  have  been  i-egarded  by  Behrens  as  fecundation  stages,  the  joint 
authors  cited  above  state  with  emphasis  that  these  "  represent  either 
abnormally  developed  oospheres  or  oogonia." 

VOLVOX. 

Without  implying  any  relationship  whatever  between  the  two  groups 
of  plants  to  which  they  belong,  the  sexual  process  in  Volvox  may  be 
fittingly  mentioned  along  with  that  of  Fucus.  In  this  most  highly 
differentiated  representative  of  the  Volvocacece  we  have  highly  special- 
ized sexual  cells,  and  in  fact,  as  has  been  already  stated  in  a  preceding 
chapter,  there  is  in  this  group  of  plants,  as  in  the  brown  algae,  a 
gradual  transition  from  the  simplest  form  of  sexual  reproduction  of 
isogametes  to  that  of  the  well  differentiated  bisexual  elements  of  Volvox. 

Some  authors  (Strasburger,  '92,  1900;  Overton,  '89)  regard  the 
spermatozoid  of  Volvox  as  a  transition  between  the  motile  isogametes 
of  algae  and  the  spermatozoids  of  the  Characeae.  The  spermatozoid 
of  Volvox  globator  tapers  gradually  to  a  slender  anterior  end  which  is 
colorless,  the  thicker  posterior  end  being  yellowish.  At  the  boundary 
between  the  two  lies  the  red  eye-spot,  and  a  little  farther  forward  are 
borne  the  two  laterally  inserted  cilia.  It  is  reasonable  to  assume  that 
the  cilia  spring  from  a  blepharoplast,  although  positive  proof  is  still 
wanting.  Strasburger  (1900,  p.  196)  regards  the  colorless  and  slender 
anterior  end  as  the  homolog  of  the  mouth-piece  of  algal  gametes,  from 
which  such  highly  differentiated  bisexual  elements  as  those  of  Volvox 


CEDOGONIUM.  go 

have  been  evolved  ;  but  in  Volvox  the  insertion  of  the  cilia  has  under- 
gone a  lateral  displacement,  so  that  they  nov^r  spring  from  the  base  of 
the  mouth-piece. 

The  large  egg-cells,  although  not  escaping  from  the  mother  colony 
into  the  surrounding  water  before  fecundation,  are  in  a  measure  free 
to  move  passively  w^ithin  the  mother  colony.  The  same  kind  of 
stimulus  operative  in  bringing  the  eggs  and  spermatozoids  together  in 
Fucus  may  in  all  probability  obtain  also  in  Volvox.  In  the  case  of 
dioecious  forms  especially,  investigation  along  this  line  will  probably 
yield  important  results,  and  with  modern  technique  a  careful  study  of 
the  behavior  of  the  sexual  nuclei  and  other  cytological  details  of  fecun- 
dation, concerning  which  we  know  practically  nothing,  will  also  bring 
to  light  much  of  value  and  interest  to  our  knowledge  of  fecundation. 

CEDOGONIUM. 

We  shall  now  pass  to  the  consideration  of  the  sexual  process  in 
certain  of  those  fresh-water  algae  in  which  the  female  gamete  remains 
enclosed  in  its  more  specialized  and  characteristic  organ,  the  oogonium. 

Beginning  with  such  forms  as  Cylindrocapsa  and  CEdogonium  we 
have  a  progressive  series  of  forms  culminating  in  Coleochcete^  in  which, 
apart  from  the  specialized  bisexual  products,  there  are  more  highly 
differentiated  and  characteristic  sexual  organs. 

The  nature  and  development  of  the  sexual  organs  in  CEdogonium 
and  the  process  of  fecundation  have  been  carefully  described  by  Pring- 
sheim  ('56)  and  others  in  so  far  as  these  phenomena  may  be  followed 
with  accuracy  in  the  living  material,  but,  as  regards  the  more  minute 
structure  of  the  spermatozoid  and  egg-cell  and  the  behavior  of  the 
sexual  nuclei  in  fecundation,  the  researches  of  earlier  observers  leave 
much  to  be  desired.  In  more  recent  years  Klebahn  ('91)  has  suc- 
ceeded in  filling  in  many  of  the  gaps,  and  it  is  to  his  investigations 
that  we  are  chiefly  indebted  for  a  more  detailed  knowledge  of  the 
behavior  of  the  nuclei. 

When  the  oogonium  {^CEdogonium  boscii)  has  attained  its  defini- 
tive form,  the  protoplasm,  which  encloses  a  large  vacuole,  is  every- 
where closely  applied  to  the  cell-wall.  Changes  which  lead  to  the 
formation  of  the  opening  in  the  upper  part  of  the  organ  are  then 
manifested.  Near  the  spot  at  which  the  oogonium  will  open  a  small 
elliptical  lamella  is  formed,  which  gives  a  cellulose  reaction.  The 
formation  of  the  lamella  proceeds  from  a  colorless  portion  of  the  cyto- 
plasm, which  can  not  be  distinguished  at  an  earlier  stage.  Between 
cell-wall  and  lamella  a  lens-shaped  cavity  arises,  and  a  transverse  slit 


90 


FECUNDATION  ;  HETEROGAMETES. 


is  formed  in  the  wall  ^ig.  31,  B).  Both  cavity  and  slit  are  probably 
the  result  of  a  swelling  of  the  wall  on  the  side  toward  the  lamella. 
The  two  edges  of  the  slit  roll  upward  and  downward  respectively,  and 
in  this  way  an  opening  is  formed  in  the  cell-wall.  The  next  stage  in 
development  is  marked  by  the  contraction  and  rounding  up  of  the 
protoplasm  to  form  the  egg,  but  the  oogonium  is  still  closed  by  the 
lamella.  The  nucleus  lies  in  the  upper  end  of  the  egg,  and  below  it 
is  the  vacuole,  which  has  become  smaller.  The  nucleus  resembles  the 
nuclei   of   the  vegetative   cells,   being    relatively   large    with   a   large 


Fig.  31. — Fecundation  in  CEdogoniutH  boscit. — ( After  Klebahn.) 


A,  spermatozoid. 

B,  young  oogonium,  showing  origin  of  opening  in  the  wall  and  lamella  beneath. 

C,  oogonium  just  after  opening. 

D-G,  upperportionsof  fecundated  eggs,  showing  successive  stages  in  fusion  of  nuclei. 

nucleolus  (Fig.  31,  C).  The  so-called  receptive  spot  near  the  upper 
end  of  the  e.gg  is  formed,  according  to  Klebahn,  by  the  withdrawal  of 
the  chloroplasts  and  not  by  the  collecting  of  a  special  mass  of  cyto- 
plasm. Finally,  the  closing  lamella  disappears  (probably  by  being 
partly  dissolved  in  water),  forming  an  opening  for  the  entrance  of  the 
spermatozoids  (Fig.  31,  C,  b).  No  part  of  the  plasmic  contents  of  the 
egg  is  expelled  on  the  opening  of  the  oogonium,  as  has  been  claimed 
by  some  observers.     That  which  is  expelled,  to  judge  from  Klebahn's 


COLEOCH^TE.  9I 

figure,  consists  merely  of  the  liquified  or  gelatinized  remains  of  the 
lamella. 

The  spermatozoid,  contrary  to  male  gametes  among  the  algae,  bears 
a  circle  of  cilia  at  its  anterior  end  (Fig.  31,  A).  It  is  not  known 
whether  the  cilia  are  developed  from  a  distinct  body  or  blepharoplast, 
or  whether  the  cilia-bearer  is  only  a  thickening  of  the  plasma  mem- 
brane, as  Strasburger  maintains  for  the  asexual  swarm-spore  of  this 
genus.  Near  the  posterior  end  of  the  spermatozoid  lies  its  small  and 
dense  nucleus,  in  which  a  nucleolus  is  not  to  be  recognized. 

Soon  after  the  spermatozoid  enters  the  egg,  probably  at  the  receptive 
spot,  its  nucleus  wanders  toward  the  egg-nucleus  (Fig.  31,  D,  E,  F). 
Before  the  final  fusion  of  the  two  nuclei,  that  of  the  spermatozoid 
increases  somewhat  in  size  (from  4/jt  to  6fx)  and  becomes  looser  in 
structure,  but  a  nucleolus  was  not  seen  in  it.  After  fusion  has  taken 
place,  the  fact  can  be  readily  recognized  in  that  the  chromatin  elements 
of  the  male  nucleus  are  distinguishable  in  the  egg-nucleus.  Very 
soon,  however,  this  characteristic  disappears;  the  male  chromatin 
granules  become  distributed  beyond  recognition  among  those  of  the 
egg-nucleus,  since  both  nuclei  are  in  the  resting  condition. 

COLEOCHiETE, 

Coleochaete  demands  a  special  consideration  not  only  on  account 
of  the  peculiarity  of  the  sexual  organs  but  also  because  this  remarkable 
plant,  owing  to  the  behavior  of  the  oosphere  subsequent  to  fecundation, 
may  be  regarded  as  a  phylogenetic  guide-post,  which  enables  us  to 
connect  with  each  other  different  groups  of  thallophytes,  and  which 
indicates  the  probable  course  traversed  by  the  ancestors  of  the  lower 
archegoniates. 

The  recent  studies  of  Jost  ('95)  and  especially  those  of  Oltmanns 
('98)  have  confirmed  the  classical  account  of  Pringsheim  ('58,  '60) 
with  the  addition  of  clearing  up  certain  obscure  cytological  details, 
which  was  possible  only  with  the  aid  of  more  improved  technique. 

In  the  development  of  the  antheridium  a  small  protuberance  is 
formed  from  the  end  cell  of  a  filament,  into  which  passes  a  daughter- 
nucleus  resulting  from  the  division  of  the  nucleus  of  the  mother-cell, 
and  which  is  cut  off  by  a  wall  formed  at  the  junction  of  the  protuber- 
ance and  the  mother-cell  (Fig.  32,  A).  No  part  of  the  chloroplast  of 
the  mother-cell  passes  into  the  antheridium.  In  addition  to  this  central 
antheridium,  others  will  be  formed  from  the  mother-cell  in  like  manner, 
so  that  finally  several  antheridia  stand  side  by  side  at  the  end  of  the 
mother-cell  as  so  many  branches  (Fig.  32,  B).     The  spermatozoids, 


92 


FECUNDATION  ;  HETEROGAMETES. 


of  which  only  one  is  borne  in  each  antheridium,  are,  according  to 
Pringsheim  ('58,  p.  297),  almost  entirely  colorless,  with  but  a  faint 
greenish  hue ;  each  bears  at  the  anterior  end  two  cilia,  one  extending 
backward  during  the  progressive  motion  of  the  cell.  In  the  absence 
of  a  chromatophore  the  spermatozoid  of  Coleochcete  differs  from  that 
of  CEdogonium^  in  which  the  chlorophyll  undergoes  a  transformation 
in  the  male  gametes,  and  in  this  respect  it  foreshadows  the  develop- 
ment of  the  sperm  in  higher  plants. 

The  oogonium  is  also  developed  from  the  end  cell  of  a  branch. 
It  is  recognized  first  by  the  presence  of  a  beak  at  the  distal  end  of  the 

cell,  which  soon  becomes  the 
neck  of  the  flask-shaped  organ 
(Fig.  32,  C,  D).  In  the  neck 
dense  coloi'less  cytoplasm  accu- 
mulates which  contains  one  or 
more  large  vacuoles.  In  the 
basal  or  ventral  portion  are  sit- 
uated the  nucleus,  a  large  vac- 
uole, and  a  laterally  placed 
chloroplast.  The  neck  now 
increases  in  length  with  an  ap- 
parent increase  in  the  quantity 
of  its  cytoplasm,  the  ventral 
portion  remaining  unchanged. 
As  soon,  however,  as  the  neck 

Fig.  32.— Development  of  sexual  organs  in  Coleochaie    hag  reached  itS  definitive  size,  3 
pulvinata. — (After  Oltmanns.)  .  .  ,  ,  . 

A,  B,  development  of  antheridium.  transformation  takcs   place   m 

c,D.  two  young  stages  of  the  oogonium.  t]^e  ventral  part  of   the  oogo- 

nium ;  the  chloroplast  leaves  its  lateral  position,  passes  down  and  applies 
itself  closely  to  the  bottom  of  the  organ  (Fig.  33,  E).  It  has  increased 
appreciably  in  size  and  contains  two  pyrenoids.  The  oogonium  opens 
probably  by  the  gelatinization  of  the  end  wall  of  the  neck.  As  soon 
as  the  organ  opens  the  cytoplasm  contracts  into  the  basal  portion  to  form 
the  egg-cell.  Whether  a  part  of  the  cytoplasm  in  the  neck  is  thrown 
off  cannot  be  stated  positively,  but  there  is  no  reason  to  believe  that 
this  occurs.  Both  Jost  and  Oltmanns  accord  in  the  opinion  that  no 
cytoplasm  is  expelled  when  the  oogonium  opens,  while  Pringsheim 
speaks  of  the  extrusion  of  a  colorless  substance  only,  which  disoi'- 
ganizes  at  once.  The  expulsion  of  a  small  quantity  of  mucilaginous 
substance,  or  even  cytoplasm,  is  utterly  without  important  significance, 
as  the  nucleus  of  the  oogonium  does  not  divide  previously  to  fecunda- 


COLEOCH^TE. 


93 


tion.  In  the  withdrawal  of  the  chloroplast  into  the  base  of  the  egg- 
cell,  and  the  formation  of  a  receptive  spot,  Coleochcete  is  paralleled 
by  both  CEdogonium  and  Vaucheria. 

Soon  after  entering  the  oogonium  the  spermatozoid  penetrates  the 
egg,  a  membrane  is  formed  about  the  latter,  and  the  sperm-nucleus 
wanders  toward  that  of  the  egg  (Fig.  33,  F).  Before  final  fusion 
takes  place,  one  or  more  changes  occur  in  the  egg,  which  may  be 
worth  noting.     The  chloroplast  which  lay  at  the  bottom  of  the  egg, 


Fig.  33. — Fecundation  in  CoUochate  pulvinata. — (After  Oltmanns.) 

E,  mature  oogonium,  egg  rounded  off. 

F-H,  oogonia  with  fecundated  eggs;  male  nucleus  in  F  applied  to  that  of  egg;  both  nuclei 

in  resting  stage. 
G,  a  little  later  than  F  ;  the  chloroplast  has  taken  a  lateral  position  in  egg. 
H,  fusion  of  sexual  nuclei  complete. 

as  previously  stated,  divides,  and  the  two  resulting  chloroplasts  take 
positions  on  opposite  sides  of  the  egg  (Fig.  33,  G).  The  egg  and, 
consequently,  the  ventral  part  of  the  oogonium  increase  in  size ;  in  the 
former  vacuoles  appear,  and  the  nuclei  which  are  in  the  resting  con- 
dition fuse  completely  (Fig.  33,  H). 

For  the  further  behavior  of  the  oospore  and  its  germination,  which, 
as  is  well  known,  bears  a  tolerably  close  resemblance  to  such  liver- 
worts as  Riccia^  the  reader  is  referred  to  the  original  papers  of 
Pringsheim  and  Oltmanns. 


94  FECUNDATION  ;    HETEROGAMETES. 


VAUCHERIA. 

With  the  possible  exception  of  Sphceroplea  annulina  var,  braunii^ 
we  have  dealt  thus  far  with  heterogamous  fecundation  in  those  algae 
with  uninucleate  cells.  We  shall  now  examine  the  sexual  process  in 
three  notable  types,  one  from  among  the  algae  and  two  from  the  fungi, 
namely,  Vaucheria^  Albugo  {Cyst opus) ^  and  Achlya^  in  which  the 
cells  are  multinucleate. 

In  the  species  under  consideration,  Vaucheria  clavata^  both  anthe- 
ridia  and  oogonia  may  be  considered  as  short  side  branches  cut  off 
from  the  parent  filament  by  transverse  septa.  The  primordium  of  the 
antheridium  (Oltmann's,  '95)  contains  numerous  small  nuclei  which 
probably  multiply  by  division.  After  the  formation  of  the  transverse 
wall,  the  nuclei  become  spindle-shaped,  move  into  the  central  vacuole, 
and  assume  a  radial  arrangement.  Each  spindle-shaped  body  sur- 
rounded by  a  court  of  fine  cytoplasm  free  from  chlorophyll  represents 
a  spermatozoid.  Very  fine  threads  visible  in  the  antheridium  were 
regarded  as  cilia. 

Concerning  the  r61e  of  the  nuclei  during  the  development  of  the 
oogonium,  the  several  authors  differ  somewhat.  According  to  Schmitz 
('79)  the  numerous  nuclei  pi'esent  in  the  young  oogonium  probably 
fuse  later  into  one.  Similar  results  were  obtained  by  Behrens  ('90). 
Schmitz  ('83)  claimed  that,  in  the  plasmic  mass  extruded  on  the 
opening  of  the  oogonium,  small  nuclear  fragments  were  present, 
which  had  probably  become  separated  from  the  nuclei  of  the  young 
oogonium.  Klebahn  ('92)  disputed  the  above  conclusions  and  asserted 
that,  long  after  fecundation,  he  had  observed  numerous  nuclei  in  each 
oospore.  Oltmanns  ('95),  using  more  exact  methods,  found  that  a 
union  of  the  several  nuclei  in  the  young  oogonium  does  not  take  place, 
but,  on  the  contrary,  all  save  one  pass  back  into  the  parent  filament 
before  the  formation  of  the  transverse  wall   cutting  off   the  oogonium. 

The  development  of  the  oogonium,  according  to  Oltmanns,  is  as 
follows :  Together  with  the  protoplasmic  mass  numerous  nuclei  pass 
into  the  primordium  of  the  oogonium  (Oogonanlage)  (Fig.  34,  A). 
The  nuclei,  which  are  in  the  neighborhood  of  the  future  beak,  prob- 
ably undergo  division,  thereby  increasing  their  number.  As  soon  as 
the  oogonium  has  reached  its  definite  size,  a  retreating  movement  of 
the  plasmic  mass  sets  in,  and  a  portion  of  the  plasma,  with  numerous 
chloroplasts  and  nuclei,  re-enters  the  mother-filament  (Fig.  34,  B). 
The  single  nucleus  remaining  tarries  awhile  in  the  beak  at  the  bound- 
ary between  the  colorless  and  chlorophyll-bearing  plasma,  but  finally 


VAUCHERIA. 


95 


it  wanders  toward  the  center  of  the  oogonium  (Fig.  34,  C),  which  is  now 
separated  from  the  filament  by  a  cross- wall.  The  egg-nucleus  retains 
this  position  until  fecundation  (Fig.  34,  D)  ;  it  does  not  divide  and  the 
probability  of  any  nuclear  substance  being  thrown  off  with  the  extru- 
sion of  a  small  plasmic  or  mucilaginous  mass  when  the  oogonium 
opens  is,  therefore,  excluded.  Although  Oltmanns  observed  in  the 
cytoplasm  of  the  beak  granules  staining  somewhat  more  intensely  than 


Fig.  34. — Fecundation  in  Vauchtria  clavata, — (After  Oltmanns.) 

A,  B,  young  oogonia  before  being  delimited  by  transverse  walls  from  filament.     In  B  all 
nuclei  save  one  are  passing  back  into  filament. 

C,  oogonium  ready  for  fecundation. 

D,  the  spermatozoid  has  entered  egg. 

E,  F,  sexual  nuclei  in  contact ;  in  F  the  male  nucleus  has  increased  in  size. 
G,  a  fusion  nucleus. 

H,  oogonium  containing  oospore  several  weeks  old. 

the  rest,  yet  he  does  not  think  it  probable  that  these  sustain  any  rela 
tion  to  the  nuclei.  At  the  upper  end  of  the  Q^'g  is  the  rather  large  recep- 
tive spot  formed  by  the  withdrawal  of  the  chloroplasts  from  that  region. 
Immediately  on  entering  the  cytoplasm  of  the  egg  the  sperm-nucleus 
increases  noticeably  in  size ;  its  linin  net,  now  more  loosely  arranged, 
reveals  many  strongly-staining  granules  which  are  probably  chromatin. 
In  the  meantime  the  egg-nucleus  enlarges  considerably,  and  appears 


^6  FECUNDATION  ;  HETEROGAMETES. 

more  distinctly  granular.  It  contains  also  a  rather  large  and  distinct 
nucleolus.  When  the  two  nuclei  come  in  contact,  the  male  is  smaller 
than  the  female  (Fig.  34,  E).  Fusion  now  takes  place  (Fig.  34,  F,  G), 
and  the  fusion-nucleus  presents  at  first  a  fine  hollow  framework  in 
which  lie  numerous  chromatin  granules  of  about  equal  size ;  later  it 
becomes  smaller  and  denser,  appearing  more  finely  granular,  when 
finally  a  large  nucleolar  body  is  again  present  (Fig.  34,  H). 

ALBUGO  (CYSTOPUS). 

The  nuclear  behavior  and  certain  cytoplasmic  phenomena  manifested 
in  the  development  of  the  sexual  organs,  especially  the  oogonium,  of 
the  genus  Albugo  is,  so  far  as  known,  unique  among  the  Thallophyta, 
if  not  in  the  plant  kingdom.  The  union  of  several  male  with  several 
female  nuclei  in  the  oosphere  of  A.  b lit i and  A.  portulacece  (Stevens, 
'99,  '01)  is  paralleled  among  plants  only  by  Pyronema  (see  p.  iii) 
and  the  possible  case  of  Sporodinia  grandis.  We  shall  confine  our- 
selves first  to  the  development  of  the  sexual  organs  and  fecundation  in 
Albugo  Candida^  referring  in  a  later  paragraph  to  the  phenomena 
described  for  A.  bliti^  A.  portulacece  and  other  closely  related  repre- 
sentatives of  the  group. 

The  following  statements  are  based  largely  upon  the  researches  of 
Wager  ('96),  probably  the  most  complete  account  published  for  this 
species.  The  observations  of  Wager  have  been  confirmed  by  the  later 
studies  of  Berlese  ('98),  Davis  (1900)  and  Stevens  ('01),  those  of  Davis 
and  Stevens  presenting  more  clearly  certain  details  regarding  the 
central  body  of  differentiated  cytoplasm  in  the  oogonium.  The  more 
obvious  details  in  the  development  of  the  sexual  organs  are  too  well 
known  to  bear  repetition,  and  consequently  the  reader's  knowledge  of 
that  part  of  the  process  is  assumed. 

The  antheridium,  which  appears  almost  simultaneously  with  the 
oogonium,  is  more  or  less  densely  filled  with  granular  cytoplasm  in 
which  several  nuclei  are  present  when  the  partition  wall  is  formed 
delimiting  the  antheridium  from  the  parent  hypha.  Previously  to 
or  during  the  early  development  of  the  conjugation-tube,  the  nuclei 
undergo  a  karyokinetic  division  by  which  their  number  is  doubled 
(Fig.  35,  A). 

When  a  quantity  of  cytoplasm  and  numerous  nuclei  have  passed 
into  the  enlarging  primordium  of  the  oogonium,  a  transverse  wall  is 
formed  separating  it  from  the  parent  hypha.  The  cytoplasm  shows  a 
foam  structure,  and  the  nuclei  are  more  or  less  regularly  spaced  in  its 
reticulum    (Fig.  35,  B).     The   nuclei   possess  a   membrane,   and  in 


ALBUGO    (CYSTOPUS). 


97 


structure  seem  not  unlike  those  of  higher  plants.  The  number  of 
nuclei  in  the  young  oogonium,  at  this  stage,  varies  with  its  size,  the 
average  being  from  70  to   no.     The  antheridium,  containing  from  6 


Fig.  35.— Development  of  sexual  organs  and  fecundation  in  Albugo  (Cystopus)  candtWa.— (After  Wjiger.) 

A,  antheridium  attached  to  wall  of  oogonium,  just  beginning  to  push  out  its  conjugating  tube;  dense 

mass  of  cytoplasm  and  several  nuclei  seen  near  projection. 

B,  young  oogonium  after  its  delimination  from  mycelium,  with  antheridium  attached  ;  receptive  papilla 
projects  from  oogonium  toward  antheridium  ;  nuclei  seem  to  be  entering  prophase  of  division. 

C,  later  stage ;  protoplasm  has  contracted  into  a  large  central  mass ;  nearly  all  the  nuclei  have  divided, 

and  are  collecting  at  periphery  of  central  mass  ;  the  deeply  stained  mass  of  cytoplasm,  a  (central  body, 
coenocentrum),  is  seen  in  center  in  contact  with  egg-nucleus;  egg-nucleus  is  derived  from  one  of  th« 
original  nuclei  of  oogonium. 

D,  o6gonium   into  which  conjugating  tube  has  penetrated ;   differentiation  of  periplasm  and  ooplasm 

becoming  apparent,  though  a  plasma  membrane  has  not  been  formed  around  the  egg;  in  center  of 
ooplasm  is  the  egg-nucleus  near  the  dense  mass  of  cytoplasm ;  in  end  of  conjugating-tube  is  dense 
cytoplasm  in  which  lies  the  male  nucleus. 

E,  later  stage  than  D ;  apical  wall  of  conjugating  tube,  becoming  very  thin ;  plasma  membrane  of  egg 

not  yet  formed. 

to  12  nuclei,  now  applies  itself  to  the  oogonium.  The  structure 
both  of  its  nuclei  and  cytoplasm  is  similar  to  that  of  the  oogonium. 
Soon  after  the  two  organs  come  into  contact  with  each  other,  a  portion 


^8  FECUNDATION  ;  HETEROGAMETES. 

of  the  C3^oplasm  just  beneath  the  wall  of  the  oogonium  on  the  side 
nearest  the  antheridium  presents  a  granular  and  more  homogeneous 
appearance.  At  this  place  a  papilla  with  a  deeply  stained  apical  spot 
is  formed,  which  tends  to  bore  its  way  through  the  wall  of  the 
oogonium,  causing  the  wall  to  become  thinner.  This  is  called  the 
receptive  papilla,  since  it  marks  the  spot  at  which  the  conjugation-tube 
penetrates  the  oogonium.  It  doubtless  facilitates  the  development  of 
the  conjugation-tube. 

In  A.  portulacece  (Stevens,  '99)  this  receptive  papilla  seems  to  pene- 
trate the  antheridium. 

The  differentiation  of  the  oospore,  which  now  begins,  is  manifested 
in  the  contraction  of  the  protoplasm  toward  the  center  into  a  rounded 
mass  connected  with  the  wall  of  the  oogonium  by  thick  plasmic 
strands.  This  mass  contains  all  the  nuclei  (Fig.  35,  C).  It  gradually 
becomes  further  differentiated  into  a  central  vacuolate  and  reticulate 
mass,  the  ooplasm,  which  becomes  the  egg-cell  or  oosphere,  and  an 
exterior  layer  of  very  dense  non- vacuolate  cytoplasm,  the  periplasm. 
With  the  exception  of  a  few  plasmic  strands,  which  extend  to  the  wall 
of  the  oogonium,  the  entire  protoplasmic  contents  outside  the  oosphere 
become  finally  condensed  into  periplasm.  The  nuclei,  located  mostly 
in  the  periplasm  and  gradually  becoming  more  and  more  restricted  to 
this  layer,  now  undergo  karyokinetic  division  whereby  their  number  is 
doubled.  Stevens  claims  that  two  mitoses  occur  in  both  sexual  organs 
during  their  development. 

While  nuclear  division  is  taking  place  a  dense  granular  and  rather 
sharply  defined  mass  of  cytoplasm  appears  in  the  center  of  the  not  yet 
completely  differentiated  oosphere  (Fig.  35,  C,  a).     Wager,  '96,  says  : 

It  is  of  the  same  nature  as  the  dense  protoplasmic  mass  which  appears  in  the 
fertilizing  tube  at  the  moment  when  it  begins  to  grow,  and  is  produced  probably 
by  an  accumulation  of  stainable  granules  from  the  protoplasm.  This  dense 
mass  of  protoplasm  can  be  observed  in  oogonia  of  all  stages,  such  as  are  figuted 
in  (1.  c.)  Figs.  8  and  22.  Shortly  after  its  appearance  one  of  the  nuclei  produced 
by  the  division  in  the  oogonium  comes  into  close  contact  with  it,  and  gradually 
becomes  more  or  less  completely  embedded  in  it.  All  the  other  nuclei  pass  to 
the  periplasm,  leaving  this  single  nucleus  in  the  center  as  the  nucleus  of  the 
ovum  (Fig.  35,  D,  E). 

At  this  stage  the  oosphere  may  be  considered  as  differentiated, 
although  its  limiting  plasma  membrane  has  not  yet  appeared. 

It  seems  that  this  central  cytoplasmic  body  or  mass  which  has 
received  much  attention  at  the  hands  of  later  observers  was  described 
by  Dangeard  as  an  oil  globule,  and  mistaken  by  Chmielewskij  for  a 


ALBUGO     ( CYSTOPUS)  .  59 

nucleus.  Swingle  ('98)  called  attention  to  this  body  in  A.  Candida^ 
which  he  was  inclined  to  regard  as  an  organ  of  the  oogonium,  taking 
some  part  in  the  delimination  of  the  egg  and  the  fusion  of  the  male 
and  female  nuclei.  A  similar  body  has  been  observed  in  A.  blitiy  A. 
tragopogonis^  and  A.  fortulacece^  by  Stevens  ('99),  who  proposed 
for  it  the  name  "  coenocentrum."  In  A.  bliti^  in  which  it  was 
described  as  structureless  and  unchanging,  this  body  does  not  seem  to 
be  so  intimately  associated  with  the  sexual  nuclei  as  in  ^.  Candida^  as 
noted  by  Wager  and  Davis.  In  A.  tragopogonis  it  occupies  an  interme- 
diate position  in  size  between  that  in  A.  bliti  and  A.  Candida^  where 
it  is  largest.  According  to  Davis's  figures  the  female  nucleus  does 
not  become  embedded  in  the  body  in  question.  In  A.  Candida  this 
body  disappears  during  the  union  of  the  sexual  nuclei  or  a  little  later. 

There  is  no  doubt  that  these  observers  refer  to  the  same  phenome- 
non, which  is  the  expression  of  a  specialized  and  tolerably  well  differ- 
entiated portion  of  the  cytoplasm  of  the  oogonium.  It  may  have  to  do 
in  some  way  with  the  delimination  of  the  egg-cell  and,  possibly,  with 
the  union  of  the  sexual  nuclei,  but  it  certainly  can  not  be  regarded  as 
an  organ  of  the  cell  or  of  the  oogonium  with  morphological  rank. 
Stevens  ('01)  regards  this  body  as  nutritive  in  character  and  exerting 
a  chemotactic  stimulus  upon  the  sexual  nuclei. 

During  the  changes  just  described  the  nuclei  of  the  antheridium  have 
been  undergoing  division,  and  their  number  is  now  about  twice  that  at 
the  beginning.  The  conjugation-tube  has  grown  and  pushed  its  way 
through  the  periplasm  into  the  plasma  of  the  ^^'g.  A  single  nucleus 
and  a  small  quantity  of  densely  staining  cytoplasm  pass  from  the 
antheridium  into  the  conjugation-tube  to  its  apex  (Fig.  35,  D).  The 
tube  now  grows  toward  the  centei  of  the  oosphere,  around  which  a 
plasma  membrane  has  not  yet  been  formed  (Fig.  35,  E).  The  dense 
mass  of  cytoplasm  in  the  end  of  the  tube  becomes  reduced  in  amount, 
having  been  used  up  probably  to  form  the  new  growing  wall  (Wager, 
'96,  p.  330).  The  growth  of  the  conjugation-tube  continues  until  it 
comes  into  contact  with  the  central  mass  of  dense  cytoplasm  (cceno- 
centrum)  referred  to  in  the  preceding  paragraphs.  As  soon  as  the  end 
of  the  tube  comes  into  contact  with  the  nucleus  of  the  egg  the  male 
nucleus  is  expelled  and  the  tube  immediately  contracts,  or  rather  col- 
lapses, and  is  withdrawn  from  or  absorbed  by  the  oosphere,  leaving  a 
large  vacuole  to  mark  its  position  (Fig.  36,  F,  a).  The  two  nuclei  are 
thus  left  in  close  contact  with  each  other,  the  male  being  slightly  smaller 
than  the  female  (Fig.  36,  F).  A  delicate  membrane,  the  plasma 
membrane,  now  becomes  visible  around  the  oosphere,  separating  it 
from  the  dense  sunounding  cytoplasm,  the  periplasm.     From  Davis's 


lOO 


FECUNDATION  ;  HETEROGAMETES. 


Fig.  5  (1.  c,  1900)  it  seems  that  the  plasma  membrane  might  be  formed 
at  an  earlier  stage.  The  sexual  nuclei  remain  close,  side  by  side,  for  a 
short  time,  and  then  fuse  to  form  the  nucleus  of  the  oospore  or  fecun- 
dated egg  (Fig.  36,  G). 

It  will  thus  be  seen  that  while  the  antheridium  of  Albugo  Candida 
contains  several  nuclei,  only  one,  together  with  a  small  portion  of 
cytoplasm,  passes  into  the  egg.  The  egg,  although  differentiated  within 
a  multinucleate  organ,  contains  but  one  nucleus,  and  fecundation  con- 
sists essentially  of  the  union  of  one  male  with  one  female  nucleus. 


Fig.  36. —  Fusion  of  sexual  nuclei  and  a  young  odspore  of 
Albugo  (Cystopus)  cemdida. — (After  Wager.) 

F,  the  conjugating  tube  within  the  egg  has  disappeared, 
sexual  nuclei  in  contact,  surrounded  by  dense  mass  of 
cytoplasm;  egg  provided  with  plasma  membrane;  a, 
vacuole  marking  position  of  conjugation-tube,  which 

has  disappeared. 

G,  young  oospore  with  fusion   nucleus  which  seems  to  be  in 

prophase  of  division. 

As  already  mentioned  in  a  preceding  paragraph,  a  remarkable  con- 
trast is  described  by  Stevens  as  taking  place  in  two  other  species  of 
Albugo^  namely,  A.  bltti  andi  A.  -portulacece.  In  the  last  two  species 
named  the  differentiated  egg-cell  is  multinucleate,  and,  since  several 
nuclei  enter  from  the  antheridium,  fecundation  consists  in  the  union 
of  several  male  with  several  female  nuclei  in  the  same  ^%'g.  This  is 
the  more  remarkable,  because  in  all  other  species  of  this  genus,  so  far 
as  the  author  is  aware,  and  in  other  closely  related  genera  of  the 
PeronosporecB,  fecundation  consists  in  the  union  of  one  nucleus  of 
each  sex.  In  A.  tragopogonis,  whose  mature  egg  is  uninucleate, 
Stevens  finds  that  the  oogonium  develops  in  the  same  manner  as  in 
A.  bliti^xid.  A.  portulacece^  but  it  is  reduced  to  a  uninucleate  condition 
by  the  disorganization  of  the  supernumerary  nuclei. 


ALBUGO    (CYSTOPUS).  lOI 

As  stated  in  the  foregoing,  the  process  described  for  A.  ih'it  and 
A.  -portulacece  is  paralleled  in  Pyronema ^  one  of  the  Asconiycetes. 
A  discussion  of  the  process  in  this  genus  will  form  a  part  of  the  next 
chapter. 

Fecundation  in  the  genera  Peronosfora  (Wager,  1900)  and  Pythium 
(Miyake,  '01  ;  Trow,  '01)  bears  a  close  resemblance  to  that  in  Albugo. 
In  the  several  species  investigated,  a  receptive  papilla  is  formed  by  the 
oogonium  during  its  development.  This  papilla  certainly  facilitates 
in  some  way  the  development  of  the  conjugation-tube,  which,  as  all 
the  observers  state,  is  formed  by  the  antheridium.  In  Araiospora 
pulchra^  Thaxter,  one  of  the  Leptomitacece,  in  which  the  periplasm 
is  developed  as  a  peripheral  layer  of  cells  surrounding  the  egg,  there 
is  some  evidence  which  suggests  that  possibly  the  conjugation-tube  is 
formed  by  the  oogonium.  Wager's  Fig.  4  for  Peronospora  seems  to 
lend  support  to  this  view  as  applied  to  that  genus. 

A  central  body  of  differentiated  cytoplasm  is  present  in  some  degree 
in  all  genera,  being  more  prominent,  perhaps,  in  Albugo  Candida  and 
Peronospora  parasitica.  Wager  and  Stevens  have  suggested  that 
it  is  functional  in  bringing  the  sexual  nuclei  together,  but  when  it  is 
known  that  in  Peronospora  parasitica  these  nuclei  separate  again 
some  distance  from  each  other  before  fusion,  it  is  difficult  to  under- 
stand the  necessity  of  such  a  body  unless  it  is  assumed  that  stronger 
forces  are  at  work  in  the  periplasm  which  tend  to  bring  all  nuclei  into 
that  region  and  retain  them  there,  the  central  body  exerting,  of  course, 
a  stronger  chemotactic  stimulus  upon  some  particular  nucleus  which 
becomes  the  egg-nucleus,  or,  in  case  of  several  egg-nuclei,  as  in  A. 
bliti  and  A.  portulacece,  upon  several  particular  nuclei.  During  the 
development  of  the  sexual  organs  in  the  several  species  in  question  a 
mitotic  division  of  the  nuclei  takes  place.  In  Pythium  ultimum 
(Trow,  '01)  the  nuclear  division  in  the  antheridium  may  follow  a  little 
later  than  in  the  oogonium,  thus  giving  the  impression  that  a  second 
mitosis  occurred.  The  division  in  both  organs  seems  to  be  simulta- 
neous in  Pythium  de  baryanum  and  Peronospora  parasitica.  Both 
Wager  and  Stevens  have  expressed  the  opinion  that  the  reduction  in 
the  number  of  the  chromosomes  occurs  in  the  antheridia  and  oogonia, 
but  no  decisive  evidence  is  at  hand. 

In  Albugo  Candida  the  sexual  nuclei  fuse  immediately  after  the 
entry  of  the  male  nucleus  into  the  oosphere,  and  the  same  is  true  for 
Albugo  portulacece.,  Peronospora  Jicaria.,  P.  alsinearum.,  Sind  P. 
e^usa,  according  to  Berlese.     In  Pythium  ultimum,  P.  de  baryanum, 

^  From  an  investigatioii  made  in  the  botanical  laboratory  of  Indiana  University,  by  Dr.  C.  A.  King. 


I02  fecundation;    heterogametes. 

and  Peronospora  parasitica^  fusion  is  retarded,  taking  place  only 
after  the  egg  has  developed  a  tolerably  thick  wall  about  itself.  The 
retarded  fusion  of  the  nuclei  has  already  been  pointed  out  for  Spiro- 
gyra^  Cosmarium,  Closterium^  and  Basidiobolus^  and,  as  will  be 
seen,  it  is  of  frequent  occurrence  in  the  plant  kingdom. 

ACHLYA  AND  SAPROLEGNIA. 

The  sexuality  of  the  Saprolegniacece  is,  perhaps,  one  of  the  oldest 
questions  in  botany  still  in  dispute.  The  fact  that  apogamy  obtains 
in  so  many  species  has  led  observers  to  accept  with  the  greatest  reserve 
any  affirmation  of  sexuality,  although  based  upon  observations  which, 
in  other  groups  of  plants,  would  not  be  questioned  as  positive  proof 
of  a  sexual  process. 

Pringsheim  ('57)  was  probably  the  first  to  attribute  to  any  represen- 
tative of  this  group  a  sexual  reproduction,  basing  his  conclusions  chiefly 
upon  a  study  of  Saprolegnia  monoica.  He  described  the  develop- 
ment of  the  sexual  organs,  the  penetration  of  the  oogonium  by  the 
conjugation-tubes,  and  their  growth  inward  among  the  egg-cells.  He 
stated  also  that  the  tubes  opened  and  discharged  their  contents  among 
the  eggs.  Reasoning  from  the  analogy  of  Vaucheria^  Pringsheim 
concluded  that  a  real  sexual  process  existed  in  the  species  in  question. 

Several  years  later  De  Bary  ('81)  combated  this  view,  alleging  that, 
as  he  did  not  observe  the  fusion  of  the  conjugation-tubes  with  the  egg- 
cells  {Saprolegnia  ferax  and  Achlya  polyandra) ,  no  fecundation 
took  place  and  that  apogamy  characterized  the  entire  group.  De  Bary 
made  a  careful  study  of  several  species,  keeping  pure  cultures  of  the 
same  running  for  several  years,  and  his  view,  it  is  safe  to  say,  has  been 
more  generally  accepted  by  botanists  than  that  of  Pringsheim. 

Pringsheim  continued  his  studies,  and  in  1882  brought  forth  addi- 
tional evidence  in  support  of  his  view.  He  described  and  figured  the 
fusion  of  the  conjugation-tubes  with  the  egg-cells  in  Achlya  poly andra^ 
and,  although  his  "  spermamoeba  "  were  nearly  amoeboid  parasites  and 
not  male  gametes,  as  he  persistently  maintained,  yet  his  collected 
observations  seemed  to  furnish  as  strong  evidence  in  favor  of  sexuality 
as  that  which  could  be  brought  against  it  by  his  opponents.  Since  the 
above  mentioned  publications  of  Pringsheim  and  De  Bary  the  majority 
of  observers  dealing  with  the  subject  have  leaned  toward  the  view  of 
De  Bary. 

Within  more  recent  years  the  subject  has  been  taken  up  by  Hartog 
('^9»  '95)  ^^^  Trow  ('95,  '99),  with  the  aid  of  improved  technique, 
especially  on  the  part  of  Trow.      Hartog  reaffirms  the  doctrine  of 


ACHLYA    AND    SAPROLEGNIA. 


[O3 


De  Bary,  while  Trow  brings  forward  fresh  evidence  in  behalf  of  a  real 
fecundation.  The  rapid  strides  made  in  our  knowledge  of  cytology  by 
the  application  of  better  methods  of  technique  and  skill  in  manipula- 
tion has  not  only  brought  to  light  fresh  questions  of  inquiry,  but  has 
made  possible  also  new  points  of  view.  Consequently,  the  observers 
last  mentioned  find  themselves  differing  not  merely  upon  the  old  ques- 
tion, but  upon  others  of  deep  significance  in  connection  with  the  sexual 
process. 

Following  each  of  the  two  publications  of  Trow  ('95,  '99)  has 
appeared  a  criticism  by  Hartog,  in  which  he  calls  into  question  the 
statements  of  the  former,  without,  however,  submitting  the  results  of 
any  new  observations.  As  will  be  shown  later,  the  chief  difference  of 
opinion  between  Hartog  and  Trow,  apart  from  the  main  contention, 
lies  in  the  behavior  of  the  nuclei  during  the  development  of  the 
oogonium  and  the  differentiation  of  the  eggs.  Hartog  finds  that, 
during  the  development  of  the  oogonium,  the  nuclei  fuse  in  groups  to 
form  the  functional  nuclei,  one  of  which  is  present  in  each  egg,  and 
concludes  with  De  Bary  that  no  fecundation  takes  place.  Trow  finds 
that  a  certain  number  of  the  nuclei  remains  functional — one  for  each 
egg-cell  developed — and  that  in  certain  species,  as  Saprolegnia  dioica 
and  Achlya  americana,  a  real  sexual  process  exists.  Trow  has  not 
demonstrated  beyond  all  question  that  fecundation  does  take  place  even 
in  the  species  that  seems  to  furnish  the  best  evidence,  but,  on  account 
of  the  superior  methods  used,  we  are  nevertheless  justified  in  believing 
that  his  results  afford  the  strongest  proof  that  has  ever  been  advanced 
in  favor  of  a  sexual  process,  and  stronger  than  all  of  his  recent  oppo- 
nents have  produced  to  the  contrary. 

Since  the  behavior  of  the  nuclei  is  of  prime  importance  in  the  differ- 
entiation of  the  sexual  elements,  and  as  this  is  one  of  the  chief  points 
in  controversy,  a  somewhat  detailed  account  of  the  behavior  of  the 
nuclei  during  the  development  of  the  oogonium  and  the  differentiation 
of  the  egg-cells  will  lead  the  reader  to  a  clearer  understanding  of  the 
questions  in  debate. 

The  young  oogonium  arises  as  a  globular  enlargement  at  the  end  of 
a  filament,  into  which  flows  dense  granular  cytoplasm  together  with 
a  number  of  nuclei.  With  an  increase  in  size  a  large  vacuole  appears 
in  the  base  of  the  oogonium,  and  this  vacuole  is  continuous  with  a 
cylindrical  vacuole  in  the  filament  (Fig.  37,  A) .  With  further  growth, 
which  is  rapid,  the  vacuole  becomes  very  large  and  the  cytoplasm  is 
confined  to  a  dense  wall-layer.  During  this  process  a  transverse  wall 
is  formed  delimiting  the  oogonium  from  the  filament.  The  nuclei, 
which  are  now  distributed  in  the  layer  of  cytoplasm,  divide  karyo- 


104  fecundation;    heterogametes. 

kinetically,  thereby  doubling  their  number,  which  may  be  ten  times 
greater  than  the  number  of  egg-cells  produced  in  the  oogonium. 
According  to  Trow  the  nuclei  reveal  a  structure  similar  to  that  in  the 
higher  plants.  Immediately  following  the  division  of  the  nuclei  rapid 
changes  take  place,  whose  interpretation  has  led  to  differences  of 
opinion.  In  both  Saprolegnia  and  Achlya^  according  to  Trow,  only 
as  many  nuclei  remain  functional  in  the  oogonium  as  there  are  egg- 
cells  developed,  the  supernumerary  nuclei  being  digested  immediately 
after  the  karyokinesis  mentioned  above  (Fig.  38,  B).  In  Achlya 
americana  the  appearance  of  the  supernumerary  nuclei  suggests  that 
they  may  possibly  divide  again  before  disorganization.  In  Saprolegnia 
the  same  author  states  that  some  of  the  degenerating  nuclei  do  really 
appear  to  unite  in  pairs.  Hartog,  on  the  contrary,  maintains  that  the 
diminished  number  of  nuclei  was  brought  about  by  nuclear  fusions, 
and  consequently  each  functional  nucleus  remaining  in  the  oogonium 
is  the  result  of  such  fusions.  Judging  from  what  we  now  know  of 
the  behavior  of  nuclei  in  multinucleated  sexual  organs  in  which  the 
sexual  nuclei  are  not  the  product  of  nuclear  fusions,  and  from  the 
evidence  which  Trow  has  furnished,  I  am  inclined  to  believe  that  the 
evidence  is  in  favor  of  his  conclusions,  namely,  that  the  functional 
nuclei  of  the  egg-cells  are  not  the  result  of  fusions. 

As  is  well  known  the  cytoplasm  now  begins  to  ball  up  in  masses 
which  eventually  form  the  egg-cells  (Fig.  37,  B,  C).  In  each  mass, 
as  in  the  completely  differentiated  e.%%^  only  one  functional  nucleus  is 
present.  Accompanying  or  surrounding  this  nucleus  is  a  conspicuous 
mass  of  finely  granular  cytoplasm,  which,  although  appearing  less 
highly  differentiated  than  in  certain  Peronosporece,  may  have  a  similar 
function.  The  young  egg  rapidly  becomes  spherical  and  is  provided 
at  first  with  a  plasma  membrane  only.  The  details  in  the  cytoplasmic 
differentiation  of  the  egg-cells  have  not,  as  yet,  been  critically  worked 
out,  except  in  so  far  as  that  is  possible  in  the  living  specimen  or  from 
observations  of  the  organs  in  toto.  Whether  the  balling  of  the  proto- 
plasm described  by  both  earlier  and  more  recent  observers  is  a  cleavage 
such  as  is  known  to  take  place  in  other  Phycomycetes  can  not  be 
affirmed  positively,  but  the  facts  seem  to  indicate  a  similar  cleavage 
or  a  closely  related  process  (Fig.  37,  B,  C).^ 

The  antheridia,  as  is  also  well  known,  are  developed  from  the  ends 
of  filaments  which  apply  themselves  closely  to  the  surface  of  the  oogo- 
nium  (Fig.  37,  D).     When  the  cross- wall  is  formed,  separating  the 

•  The  process  of  the  differentiation  of  the  egg-cells  as  described  in  the  foregoing  paragraph  is  con- 
firmed by  the  very  careful  observations  of  B.  M.  Davis  on  Saprolegnia  mixta  The  manuscript  of 
these  pages  had  left  my  hands  before  the  receipt  of  Professor  Davis's  paper 


ACHLYA    AND    SAPROLKGNIA. 


105 


antheridium  from  the  filament  it  contains  a  small  but  variable  number 
of  nuclei.  These  nuclei  undergo  the  same  changes  as  those  in  the 
oogonium,  i.  e.,  they  divide  karyokinetically,  and  some  disorganize. 
The  fecundation-tubes  are  now  developed  and  usually  more  than  one 
from  each  antheridium.     They  penetrate  the  wall  of  the  oogonium  at 


^e.ati.S 


Fig.  37. — Stages  in  development  of  sexual  organs  of  A.  ameri- 
cana  var.  cxmbrica. — (After  Trow.) 

A,  young  oogonium  before  delimination  from  hypha;  base 
of  globular  enlargement  incloses  a  vacuole  which  is  con- 
tinued back  into  hypha. 

B,  much  later  stage;  an  antheridium  applied  to  oogonium, 
both  organs  delimited  from  hyphae ;  protoplasm  of  oogo- 
nium, which  now  forms  a  thick  wall-layer,  has  begun  to 
ball  up  to  form  the  eggs . 

C,  still  later  stage  ;  balling  more  pronounced. 

D,  oSgonium  with  differentiated  eggs ;  conjugation-tubes  from 
applied  antheridium  have  entered  oogonium  and  become 
applied  to  some  of  the  eggs. 

A-D  drawn  from  living  material. 


^ 


the  thinner  places  or  pits,  and  grow  in  among  the  eggs  (Fig.  37,  D,  f .  t.). 
These  tubes  contain  nuclei  which  are  exactly  like  those  of  the  eggs, 
though  smaller.  In  one  case  Trow  was  able,  as  he  states  (99,  p.  159) 
to  trace  the  fecundation-tube  without  a  break  into  an  egg  which  was 
already  surrounded  by  a  delicate  membrane  (Fig.  38,  C).  This 
instance  ''suggests  that  the  fertilization-tube  grows  up  to  the  egg, 
presses  against  it,  indents  it,  stimulates  it  to  the  formation  of  a  cell-wall, 
and  grows  obliquely  into  the  mass  of  protoplasm,  carrying  at  its  apex 
a  single  nucleus  (Fig.  38,  C).  .  .  .  Later  stages  tend  to  show 
that  the  wall  of  the  tube  within  the  oosphere  breaks  down,  the  nucleus, 


io6 


FECUNDATION  ;  HKTEROGAMETES. 


together  with  a  small  quantity  of  protoplasm,  is  liberated,  and  so  comes 
to  lie  in  the  peripheral  part  of  the  egg.  The  cell-wall  of  the  oosphere 
is  then  completed,  and  the  end  of  the  fertilization-tube  remains  firmly 
attached  to  it."  Although  the  presence  of  the  male  nucleus,  while  in 
the  periphery  of  the  egg,  was  not  clearly  demonstrated,  yet  this  is  not 
absolute  proof  to  the  contrary.  "  I  have,"  Trow  continues,  "  satisfied 
myself,  however,  of  the  presence  of  two  nuclei  in  the  egg  a^  all  thnes 
in  this  stage,  one  peripheral  and  one  central,  and  the  peripheral  one 
always  close  to  the  point  of  attachment  of  the  fertilization-tube." 


fs-^ 


Fig.  38. — Young  stages  of  two  oOgonia  and  two  egg-cells  oi  A. 
americana  var.  cainbrica. — (After  ']  row.) 

A,  section  of  young  stage  before  delimination  from  hypha, 
showing  cytoplasmic  structure  and  nuclei. 

B,  median  section  at  stage  preceding  balling;  /.  g.  «.,  female 
gamete  nuclei ;  deg.  n.,  degenerate  nuclei. 

C,  egg  containing  one  nucleus ;  apex  of  conjugation-tube  con- 
taining male  nucleus  has  penetrated  egg. 

D,  egg  from  a  5-day  culture,  in  which  the  two  gamete  nuclei 
are  in  contact ;  m.  g.  m.,  male  gamete  nucleus. 


At  a  later  stage  obtained  from  a  five-day  culture  the  two  nuclei  are 
found  applied  to  each  other  in  the  center  of  the  egg  (Fig.  38,  D). 
They  are  in  the  resting  condition,  and  about  the  same  size,  the  male 
being  distinguished  from  the  female  only  by  its  smaller  nucleolus. 
From  the  fact  that  the  sexual  nuclei  were  found  side  by  side  in  a  five- 
day  culture,  and  from  an  examination  of  many  hundreds  of  oospores 
from  six-  to  eight-day  cultures,  it  is  inferred  that  about  three  days  are 
required  for  the  complete  fusion,  during  which  time  the  nuclei  remain 
in  the  resting  condition,  a  phenomenon  of  frequent  occurrence  among 
thallophytes.  In  the  oospores  of  nine-  or  ten-day  cultures,  which 
have  developed  a  well-differentiated  cell-wall,  only  one  nucleus  was 
observed.     Later,  during  germination,  the  fusion  nucleus  divides  karyo- 


ACHLYA   AND    SAPROLEGNIA.  IO7 

kinetically,  and  the  process  is  repeated  until  by  the  time  a  germ-tube 
is  evident,  or  even  before,  about  twenty  nuclei  are  present. 

It  may  be  objected  that  Trov^^'s  evidence  of  the  passage  of  the  sperm 
nucleus  into  the  egg  is  insufficient,  and  that  the  two  nuclei  seen  in  the 
young  oospore  may  have  been  derived  from  a  division  of  the  unfecun- 
dated  egg-nucleus.  While  such  objections  have  but  little  weight,  yet 
we  must  admit  that  the  possibility  of  their  truth  is  not  excluded.  For 
many  of  us  Trow's  observations  will  have  a  probability  bordering  on 
certainty.  Although  the  conclusions  of  Trow  require  confirmation, 
yet  I  think  it  can  be  fairly  said,  and  that  too  with  all  due  respect  for 
the  ability  and  skill  of  De  Bary  and  others  whose  observations  tend  to 
confirm  his  view,  that  Trow  has  furnished  the  strongest  evidence  that 
has  thus  far  been  brought  forward  in  support  of  the  existence  of 
sexuality  in  certain  species  of  the  Saprolegniaceae. 

From  the  foregoing  it  is  clear  that  certain  similarities  exist  between 
these  genera  and  such  forms  as  Albugo.  The  development  of  the 
sexual  organs  themselves,  and  the  earlier  conduct  of  the  numerous 
nuclei  which  enter  the  young  sexual  organs  from  the  parent  hyphae, 
are  quite  parallel.  The  great  difference  lies  in  the  differentiation  of 
the  egg-cells.  In  Safrolegnia  and  Achlya  we  have  developed,  as  a 
rule,  several  eggs,  and  there  is  no  trace  of  periplasm.  The  super- 
numerary nuclei  disoi'ganize  before  the  egg-cells  are  differentiated.  In 
Albugo  and  closely  related  genera,  the  supernumerary  nuclei,  if  we 
may  be  permitted  to  speak  of  those  of  the  periplasm  as  such,  having 
different  and  additional  functions,  disappear  later. 


CHAPTER    v.— TYPE    OF  THE   ASCOMYCETES   AND 
RHODOPHYCE^. 

Within  recent  years  our  knowledge  of  the  sexual  process  in  certain 
of  the  higher  fungi,  the  Ascomycetes,  has  been  greatly  advanced  by 
the  classical  researches  of  Harper.  These  researches  have  inaugurated 
a  sort  of  renaissance  in  the  study  of  the  sexual  process  in  the  fungi ; 
for  vi^ithin  the  last  decade  the  doctrine  of  sexuality  in  the  Ascomycetes 
as  advanced  by  De  Bary  has  been  strenuously  denied  in  some 
quarters,  especially  among  the  mycologists  of  the  Brefeldian  school, 
and  the  view  that  no  sexual  reproduction  at  all  occurs  in  this  group 
had  gained  considerable  ground. 

Harper's  work  upon  certain  Perisporeacece  and  Discomycetes  leave 
no  doubt  concerning  the  true  sexual  process  in  those  groups,  and  it  is 
reasonable  to  expect  that  further  research  will  bring  to  light  the 
presence  of  sexual  reproduction  in  other  genera  in  which  the  existence 
of  sexuality  seems  far  more  questionable. 

In  the  development  of  the  sexual  organs  and  in  the  behavior  of  the 
egg-cell,  there  is  represented  here  a  type  of  sexual  reproduction  very 
different  from  that  known  in  other  fungi  and  in  the  green  algae.  The 
closest  parallel  is  found  in  the  Rhodophycece  and  in  certain  lichens. 
There  is  certainly  a  striking  and  suggestive  resemblance  between  the 
structure  of  the  sexual  organs  and  the  process  of  development  subse- 
quent to  fecundation  in  Sphcerotheca^  Pyronema  and  Collema  on  the 
one  hand,  and  in  such  forms  of  the  red  algae  as  Batrachospertnum 
and  Nemalion  on  the  other.  It  is  not  improbable  that  further  research 
will  reveal  a  tolerably  well  connected  series  from  forms  like  Sphcero- 
theca  to  the  remarkably  complex  Dudresnya^  and  we  may  accept 
without  much  reserve  the  view  that  the  great  groups  to  which  these 
representatives  belong  represent  related  phylogenetic  series.  In  this 
chapter,  therefore,  I  shall  present  the  sexual  process  in  Sphcerotheca, 
Pyronema^  Collema^  Batrachospermutn  and  Dudresnya  as  repre- 
sentative of  the  type  of  sexuality  in  the  Ascomycetes,  including  that 
form  in  lichens,  and  in  the  Florideae. 

What  follows  concerning  Sphcerotheca  and  Pyronetna  is  based 
exclusively  upon  the  studies  of  Harper  ('95,  '96,  1900). 

SPHiEROTHECA. 

Both  antheridia  and  oogonia  of  Sphcerotheca  arise  as  lateral  branches 
of  neighboring  mycelial  filaments,  the  development  of  the  oogonium 


SPH^EROTHECA. 


109 


preceding  somewhat  that  of  the  antheridium.  Each  consists  at  first  of 
a  short  oval  branch,  which  is  distinguished  from  the  ordinary  vegeta- 
tive hyphae  only  by  its  denser  protoplasmic  contents  and  by  standing 
at  right  angles  to  the  surface  of  the  leaf  of  the  host  plant. 

As  soon  as  the  oogonium  has  attained  a  length  equal  to  two  or  three 
times  its  width,  and  a  diameter  which  is  about  twice  that  of  a  mycelial 
filament,  it  is  cut  off  from  the  parent  hypha  by  a  cross- wall.  At  this 
stage  it  possesses  a  single  nucleus  which  can  scarcely  be  distinguished 
from  the  nuclei  of  vegetative  cells.  Frequently,  before  the  young 
oogonium  is  delimited  by  the  cross- wall,  the  antheridial  branch  appears 
quite  near  the  base  of  the  former,  and  grows  upward,  closely  applied 
to  its  side  (Fig.  39,  A).  The  oogonium  appears  to  grow  faster  than 
the  antheridial  branch  at  first,  thereby  bending  over  toward  the  latter, 


E       1      / 

Fig.  39. — Sexual  organs  and  fecundation  in  Spheerothtc%  castagni  Lm. — (After  Harper.) 

A,  young  oogonial  and  antheridial  branch,  oogonium  on  left. 

B,  later  stage  of  same,  antheridium  delimited  by  a  transverse  wall. 

C,  copulation  of  antheridium  and  oogonium  ;  the  two  sexual  nuclei  in  contact  in  oogonium. 

D,  oogonium  in  which  the  sexual  nuclei  have  fused. 

E,  young  ascogonium  with  two  nuclei ;   wall  of  perithecium  is  now  several  cells  in  thickness. 


and  giving  the  impression  that  the  contiguous  walls  were  grown 
together,  and  that  the  growth  of  the  oogonium  was  retarded  on  the 
side  next  the  antheridium.  The  antheridial  branch  is  now  separated 
from  its  mycelial  filament  by  a  cross-wall  which  is  higher  in  position 
than  the  corresponding  wall  of  the  oogonium.  This  cell  contains  also 
only  one  nucleus.  When  the  development  of  the  oogonium  is  com- 
plete the  antheridial  branch  elongates  and  its  nucleus  divides.  One  of 
the  resulting  daughter-nuclei  passes  into  the  somewhat  attenuated  end 
of  the  cell,  which  is  cut  off  from  the  lower  part  to  form  the  anthe- 
ridium (Fig.  39,  B).  While  the  stalk  cell  now  elongates  and  the 
antheridium  increases  in  size  the  oogonium  experiences  little  change ; 
consequently,  the  antheridium  is  carried  upward,  and  finally  comes  to 
lie  as  a  cap  placed  more  or  less  obliquely  on  the  top  of  the  oogonium. 
At  this  stage  the  nucleus  of  the  egg-cell  is  larger  than  the  ordinary 


no 


ASCOMYCETES  AND  RHODOPHYCE^. 


vegetative  nuclei,  vv^hile  that  of  the  antheridium  is  correspondingly 
smaller. 

The  cell-walls  between  the  antheridium  and  oogonium  are  dissolved, 
the  male  nucleus  passes  through  the  opening  thus  formed  into  the 
oogonium,  wanders  toward  the  egg-nucleus,  and  soon  fuses  with  it 
(Fig.  39,  C).  After  the  entrance  of  the  male  nucleus  the  antheridium 
still  remains  filled  with  cytoplasm  which  is  in  direct  communication 
with  the  cytoplasm  of  the  oogonium.  Very  soon,  however,  the  open- 
ing between  the  two  organs  is  closed  by  a  new  wall,  when  only  a 
small  quantity  of  cytoplasm  is  to  be  seen  in  the  antheridium. 

Immediately  after  fecundation  the  oogonium  begins  a  steady  growth. 
The  egg-cell  does  not  round  off  by  means  of  self-plasmolysis  either 
before  or  after  fecundation,   thereby  becoming   separated  from  the 


Fig.  40. — Development  of  ascogonium  oi Spharotkeca  castagni. — (After  Harper.) 

F,  ascogonium  with  two  cells  ;  upper  cell  has  two  nuclei. 

G,  mature  ascogonium;  the  penultimate,  or  ascogenous  cell,  contains  two  nuclei. 

H,  the  two  nuclei   in   the  young  ascus  have  fused,  fusion  nucleus  containing  two  nucleoli. 

wall  of  the  oogonium.  In  this  respect  the  Ascomycetes  differ  from 
all  other  plants  except  the  Rhodophycece  with  which  they  form  a 
striking  parallel. 

A  few  steps  further  in  the  development  of  the  fecundated  egg  will 
be  traced  to  show  the  relation  in  the  course  of  development  of  the 
fusion  of  the  sexual  nuclei  to  the  vegetative  nuclear  fusion  occurring 
in  the  young  ascus.  In  speaking  of  this  part  of  the  development  the 
term  ascogonium  will  be  used. 

A  series  of  nuclear  and  cell-divisions  now  follow  in  the  developing 
ascogonium,  so  that  ultimately  a  row  of  five  or  six  broad  cells  result 
(Fig.  39,  D,  E,  and  Fig.  40,  F,  G).  Nuclear  and  cell-division  are 
not  dependent  upon  each  other,  and  they  do  not  seem  to  follow  in  the 
same  order.  In  different  stages  of  this  growth,  from  one  to  three 
nuclei  are  to  be  seen  in  the  distal  cell  of  the  ascogonium,  but  when  the 
definite  number  of  cells  is  formed  two  nuclei  are  always  to  be  found 


SPH^ROTHECA.  Ill 

in  the  penultimate  cell  of  the  row,  while  all  other  cells  of  the  ascogo- 
nium  are  uninuclear  (Fig.  40,  G).  This  penultimate  cell  becomes 
the  ascus  ;  it  is  not  to  be  regarded  as  the  exact  equivalent  of  any  other 
cell  of  the  ascogonium,  and  its  two  nuclei  are  not  necessarily  sister 
nuclei,  for  before  the  last  cross-wall  is  formed  in  the  ascogonium  the 
distal  cell  may  contain  three  nuclei,  and  of  these  any  pair  may  remain 
in  the  penultimate  cell.  With  further  development  these  two  nuclei 
fuse  (Fig.  40,  G,  H).  This  fusion  is  comparable  to  the  nuclear 
fusion  occurring  generally  in  young  asci,  and  consequently  it  has  not 
the  significance  of  fecundation,  but  represents  merely  a  vegetative 
union.  In  this  connection  it  may  be  mentioned  that  the  objections  which 
Dangeard  ('97)  has  raised  against  the  true  sexual  process  described 
by  Harper  do  not  seem  to  me  to  merit  any  serious  consideration. 

Sphoerotheca  represents  one  of  the  simplest  and  perhaps  the  most 
primitive  forms  of  the  true  Ascomycetes^  especially  as  regards  the 
development  of  the  ascogonium.  In  Erysiphe  and  Ascobolus  a 
greater  complexity  in  the  development  of  the  ascogonium  obtains,  but 
there  can  be  no  doubt  as  to  the  nature  of  their  sexual  organs  and  the 
fusion  of  their  true  sexual  nuclei,  especially  in  Erysiphe,^ 

PYRONEMA. 

In  Pyronetna  we  have  a  form  which  possesses  for  us  a  twofold 
interest.  I  refer  to  the  trichogyne-like  organ  borne  by  the  oogonium 
and  the  multiple  fecundation,  or  the  fusion  in  pairs  of  two  or  more 
male  with  two  or  more  female  nuclei  in  the  oogonium. 

The  development  of  the  sexual  organs  is  briefly  as  follows :  The 
cells  of  the  mycelium  from  which  these  organs  are  developed  are 
multinucleate.  Both  oogonia  and  antheridia  arise  from  the  apical 
cells  of  thick  hyphal  branches,  standing  vertical  to  the  substratum. 
The  young  oogonium  is  more  spherical  and  can  be  distinguished 
from  the  young  club-shaped  antheridium  standing  by  its  side.  Soon 
a  small  papilla  appears  at  the  apex  of  the  oogonium,  which  event- 
ually becomes  the  conjugating-tube  or  trichogyne  (Fig.  41,  A.  B). 
Both  organs  are  multinucleate  from  the  start,  the  number  of  nuclei 
increasing  by  division  as  the  cells  grow  in  size.  "The  nuclear  multi- 
plication, however,  is  out  of  proportion  to  the  vegetative  growth, 
so  that  when  the  sexual  cells  are  mature  they  contain  relatively  to 
their  size  more  nuclei  than  do  the  ordinary  vegetative  mycelial  cells  " 
(Harper,  1900,  p.  341).  A  broad  stalk-cell  is  cut  off  from  the 
base  of  the  oogonium  at  a  relatively  late  stage  in  its  development, 

»  For  a  detailed  discussion  of  these  processes  and  the  phylogenetic  significance  of  the  ascus  fruit, 
the  reader  is  referred  to  the  original  papers  of  Professor  Harper  ('95,  '96,  1900). 


112 


ASCOMYCETES    AND    RHODOPHYCE-*;. 


and  a  number  of  stalk-cells  is  usually  to  be  distinguished  at  the 
base  of  the  antheridium.  With  further  development  the  papilla  or 
young  conjugating-tube  elongates  rapidly,  its  tip  curving  somewhat 
to  meet  the  end  of  the  club-shaped  antheridium  which  curves  slightly 
over  the  oogonium,  frequently  exceeding  the  latter  in  height  (Fig. 
41,  C).  At  first  the  contents  of  the  trichogyne  and  the  oogonium  are 
continuous  (Fig.  41,  B).  It  is  multinucleate,  and  the  nuclei  do  not 
appear  to  be  different  from  those  of  the  oogonium.  Long  before  the 
trichogyne  becomes  fused  with  the  antheridium,  a  cross-wall  is  formed 


Fig.  41. — Development  of  sexual  oi^ans  in  PyronttMa  eonflntnt. — (After  Harper.) 

A,  young  pair  of  sexual  organs  with  several  vegetative  cells  below. 

B,  older  pair,  trichogyne  not  yet  delimited  from  oogonium  ;  antheridium  cut  transversely,  hence  seen 

in  transverse  section. 

C,  oogonium  and  antheridium  in  longitudinal  section  ;  oogonium  stalk  with  budding  vegetative  hyphae ; 

trichogyne  with  hyaline  beak,  its  nuclei  swollen  and  transparent. 

at  the  juncture  of  the  tube  with  the  oogonium,  delimiting  it  from  the 
latter.  This  wall  is  formed  before  the  sexual  cells  or  the  trichogyne 
have  reached  their  mature  size.  Whether  nuclear  divisions  occur  in 
the  tube  after  it  is  cut  off  was  not  determined  (Fig.  41,  C).  During 
subsequent  growth  the  nuclei  in  the  trichogyne  do  not  increase  in  size 
as  do  those  of  the  antheridium  and  oogonium,  but  sooner  or  later  show 
signs  of  disintegration.  They  swell  up  without  an  increase  of  their 
contents  until  they  may  equal  in  size  the  sexual  nuclei,  but  they  are 
very  transparent.     Later,   during  the  formation   of   the  fusion-pore 


PYRONEMA.  Ill 

between  the  trichogyae  and  the  antheridium,  these  nuclei  collapse  and 
break  down  into  dense  strands  or  shreds,  which  are  frequently  so 
connected  as  to  form  a  coarse  and  much  broken  network  in  the  cyto- 
plasm (Fig.  42,  D) .  The  structure  of  the  mature  sexual  organs,  -v^hich 
are  aggregated  in  rosette-like  clusters,  is  summarized  by  Harper  as 
follows  (1900,  p.  344)  : 

The  oogonium  is  a  spherical  or  flask-shaped  cell  filled  with  dense  protoplasm 
and  many  nuclei,  which  are  very  much  larger  than  those  of  the  ordinary  vege- 
tative cells.  Its  stalk  consists  of  two  or  three  broad  disk-shaped  cells,  of  which 
the  basal  one  is  a  part  of  the  mass  of  thickened,  swollen  cells  forming  the  base 
of  the  rosette.  The  apex  of  the  oogonium  is  continued  into  the  narrow  conju- 
gating tube  which  curves  upward  to  unite  with  the  end  of  the  antheridium.  The 
antheridium  is  a  curved,  club-shaped  cell,  thickest  near  its  upper  end,  and  taper- 
ing gradually  to  its  base,  where  it  is  continued  into  a  stalk  of  one  or  more  cells. 
The  basal  wall  of  the  antheridium  is,  as  a  rule,  somewhat  higher  up  than  that 
of  the  oogonium.  It  follows  a  somewhat  oblique  path  upward,  conforming 
rather  closely  to  the  surface  of  the  oogonium,  and  its  apex  is  even  with,  or 
reaches  somewhat  past,  that  of  the  latter. 

The  mutual  relation  of  the  sexual  organs  will  be  best  understood 
from  Fig.  44. 

The  changes  taking  place  in  the  mature  sexual  apparatus,  and  which 
lead  up  to  fecundation,  are  of  much  interest,  especially  when  compared 
with  the  same  process  in  other  plants  exhibiting  similar  phenomena. 
First  among  these  are  what  may  be  termed  the  receptive  spots  of  both 
the  antheridium  and  the  trichogyne.  In  that  part  of  the  antheridium 
near  which  the  tip  of  the  trichogyne  presses  against  its  wall  and  where 
the  fusion-pore  is  forhied,  an  area  of  protoplasm  is  differentiated  as  a 
finely  granular  and  irregularly  lens-shaped  disk  from  which  the  nuclei 
have  withdrawn.  This  granular  area,  although  situated  in  the  anthe- 
ridium. Harper  very  fittingly  compares  to  the  so-called  mouth-piece,  or 
receptive  spot,  of  the  egg  in  such  algae  as  CEdogonium  and  Vaucheria. 
The  beak-like  prolongation  of  the  trichogyne  reveals  also  a  similar, 
though  less  conspicuous,  cytoplasmic  differentiation  (Fig.  41,  C;  Fig. 
42,  D).  These  areas  seem  to  exercise  a  chemotactic  influence  which 
tends  to  bring  together  the  tube  and  the  antheridium,  and  also  to  secrete 
an  enzyme  by  which  the  walls  are  dissolved  in  the  formation  of  the 
conjugation-pore.  The  presence  of  a  similar  differentiation  in  both 
the  tube  and  the  antheridium  would  seem  to  indicate  also  that  the 
influence  is  mutual. 

At  the  point  where  the  beak  of  the  trichogyne  is  closely  pressed 
against  the  antheridium  the  walls  are  dissolved  and  a  pore  is  formed 
by  which  the  cytoplasm  of  these  two  cells  is  made  continuous  (Fig. 


114 


ASCOMVCKTES    AND    RIIODOPIIYCE.IJ. 


42,  D).  During  this  process,  or  sometimes  later,  as  stated  in  a  pre- 
ceding paragraph,  the  nuclei  of  the  trichogyne  disintegrate.  When 
this  has  taken  place  the  antheridial  nuclei  begin  to  migrate  through  the 
pore  into  the  trichogyne,  whose  protoplasmic  contents  become  still 
further  disorganized.  This  migration  of  nuclei  continues  until  the 
tube  is  quite  densely  filled  and  sometimes  slightly  swollen  (Fig.  42,  E). 
In  the  meantime  conspicuous  changes  have  been  taking  place  in  the 
oogonium.  The  nuclei,  which  are  evenly  distributed  throughout  the 
interior  of  this  organ,  begin  to  migrate  toward  the  center,  where  they 


Fig.  42. — Copulation  of  sexual  organs  of  Pyronema. — (After  Harper.) 

D,  the  trichogyne  has  copulated  with  antheridium,  nuclei  in  trichogyne  disintegrated ;  hypothecial 
hyphae  springing  from  stalk  cells. 

E,  trichogyne  filled  with  nuclei  from  antheridium  ;  antheridium  curved  around  trichogyne  so  that  the 

latter  appears  in  section  to  cut  through  it ;  trichogyne  still  separated  from  oogonium  by  transverse 
wall. 

become  collected  into  a  dense,  hollow  sphere,  equal  in  diameter  to 
about  half  that  of  the  oogonium,  or  they  may  aggregate  into  an  irreg- 
ular, crescent-shaped  mass  in  either  the  upper  or  lower  half  of  the 
oogonium  (Fig.  42,  E).  Less  frequently  several  masses  of  nuclei  are 
formed  instead  of  one. 

The  cytoplasm  of  the  oogonium,  which  was  charged  with  densely 
staining  substances,  becomes  tenuous  and  loosely  spongy  in  texture. 
After  the  oogonial  nuclei  have  aggregated  in  the  center  of  the  egg-cell, 
the  basal  wall  of  the  trichogyne  breaks  down  and  the  antheridial  nuclei 


PYRONEMA. 


»I5 


pass  at  once  into  the  oogonium,  to  the  central  mass  of  egg-nuclei,  and 
become  mingled  with  them  (Fig.  43,  F).  The  number  of  male  nuclei 
entering  the  oogonium  does  not  seem  to  be  exactly  the  same  as  the 
number  of  egg-nuclei  to  be  fecundated.  Both  sexual  organs  arise  as 
multinucleate  cells,  and,  as  there  is  no  evidence  subsequently  of  a 
parallel  series  of  nuclear  divisions  in  each,  it  is  difficult  to  see  how 
exactly  the  same  number  could  be  provided  in  each  organ. 

Only  a  small  portion  of  the  cytoplasm  of  the  antheridium  passes 
into  the  egg-cell,  so  that  here,  as  elsewhere  in  the  plant  kingdom,  the 
superior  significance  of  the  nuclei  in  fecundation  is  strikingly  mani- 
fested. The  male  and  female  nuclei  mingled  in  the  central  group  are 
indistinguishable  in  size,  structure,  and  staining  qualities,  so  that  it  is 


Fig  43. — Fusion  of  sexual  nuclei  in  the  oogonium  of  Pyr  one/Ma. —{Ahcr  Harper.) 

F,  basal  wall  of  trichogyne  dissolved,  male  and  female  nuclei  collected  in  dense  mass 

in  center  of  oogonium  and  fusing  in  pairs  ;  nuclei  still  present  in  trichogyne  alid 
upper  end  of  antheridium  ;  ascogenous  hyphse  budding  out  from  oogonium. 

G,  group  of  fusing  nuclei  from  central  mass  of  nuclei  in  an  oogonium. 

impossible  to  pick  out  a  single  nucleus  and  say  whether  it  has  come 
from  the  oogonium  or  antheridium.  The  nuclei  fuse  in  pairs  while 
they  are  aggregated  in  the  dense  mass  (Fig.  43,  G).  The  behavior 
of  all  the  nuclei  in  the  center  of  the  mass  was  not  determined  with 
certainty,  but  there  is  every  reason  to  believe  that  the  rule  of  fusion 
in  pairs  holds  for  nearly  the  whole  mass.  Harper  expressly  states  that 
there  is  no  general  fusion  of  the  nuclei  into  a  single  mass,  as  can  be 
clearly  seen  when  the  nuclei  scatter  after  fusion. 

The  oogonium  of  Pyronema  functions  at  once  as  an  ascogonium. 
All  fecundated  nuclei  pass  into  ascogenous  hyphae  and  may  reach  the 
asci.  Here  the  young  ascus  develops  also  from  the  penultimate  cell 
of  a  bent  ascogenous  hypha,  and  in  it  two  nuclei  are  present  which 


Il6  ASCOMYCETES    AND    RHODOPHYCE^. 

fuse,  but  this  fusion  does  not,  as  previously  stated  for  Sfhcerotheca^ 
represent  a  sexual  process. 

It  will  thus  be  seen  that  the  process  of  fecundation  in  Pyronema 
consists  in  the  union  of  multinucleated  gametes  and  in  the  fusion  of 
their  nuclei  respectively  in  pairs.  Here  as  in  all  other  plants,  whether 
possessing  uninuclear  or  multinuclear  gametes,  the  fact  of  prime 
importance  is  the  fusion  of  the  sexual  nuclei,  the  cytoplasm  playing 
perhaps  an  incidental  and  secondary  r61e.  The  fusion  of  numerous 
pairs  of  sexual  nuclei  in  the  egg-cell  is  after  all  not  so  remarkable  since 
the  significance  and  final  result  is  the  same  as  in  the  case  of  uninuclear 
gametes.  We  may  upon  strong  grounds  conclude  with  Harper  that 
"  this  aggregation  of  nuclei  at  the  time  of  fertilization  seems  to  be 
simply  a  provision  for  the  pairing  of  male  and  female  nuclei  with  the 


Fig.  44. — Group  of  3  pairs  of  sexual  organs  of  Pyronema  in  surface  view. — (After  Harper.) 

greatest  certainty  and  despatch."  The  cell,  considered  as  a  morpho- 
logical and  physiological  unit,  is  just  the  same  no  matter  whether  it 
possesses  one  or  many  nuclei,  and  in  this  respect  there  seems  to  be  no 
good  reason  for  regarding  a  "coenocyte  "  as  a  tissue. 

BATRACHOSPERMUM. 

As  representing  the  sexual  process  in  the  Rhodophycece  I  have 
selected  Batrachospermutn  and  Dudresnya.  Batrachospermum  is 
selected  on  account  of  the  comparative  simplicity  of  the  spore-fruit 
development,  and  because  the  fusion  of  the  sexual  nuclei,  as  observed 
by  Osterhout  (1900),  leaves  not  the  slightest  doubt  as  to  the  exact 
nature  of  a  sexual  reproduction.  The  classical  object,  Dudresnya^ 
affords  an  illustration  of  a  complex  series  of  phenomena  following 
fecundation,  which  has,  until  recently,  been  regarded  as  representing 
several  separate  sexual  acts. 


BATRACHOSPERMUM.  ifij 

With  the  process  of  fecundation  as  the  primary  object  in  view, 
Batrachospermum  has  been  recently  studied  by  Davis  ('96),  Schmidle 
('99),  and  Osterhout  (1900).  As  regards  the  cytological  details 
bearing  upon  fecundation  the  work  of  Osterhout  seems  to  have  been 
the  most  thorough. 

The  well  known  female  sexual  organ,  the  carpogonium,  of  Batra- 
chospermum^ is  a  single  cell  consisting  of  a  somewhat  flask-shaped 
basal  part,  the  trichophore,  in  which  is  the  egg-nucleus,  connected  by 
a  narrow  neck  to  the  elongated,  cylindrical  or  club-shaped,  upper 
part,  the  trichogyne  (Fig.  45,  B).  In  B.  boryanum  Sirodot,  the 
species  studied  by  Osterhout,  the  chromatophore  of  the  trichophore 
is  continued  into  the  trichogyne.  The  structure  of  the  nucleus  is 
the  same  as  that  of  higher  plants.  The  spermatia  are  globular  cells 
/^^  y^^-"-^  with  one  nucleus  and  a 

^V-iic^         H^'^ji.'%\\  reduced   chromatophore 

in  younger  stages  (Fig. 
A     45,  A). 

Fig.  45. — Sexual  organs  of  Batrachospermum  boryanutn 
Sirodot. — (After  Osterhout.) 

A,  antheridium  with  one  nucleus  and  vacuolate  cytoplasm. 

B,  mature  carpogonium  before  fecundation. 

C,  spermatium  has  copulated  with  trichogyne  of  carpogo- 
nium ;  cytoplasmic  fusion  has  taken  place,  but  nucleus 
is  still  in  spermatium ;  egg-nucleus  lies  in  tricho- 
phore. 

Schmidle  ('99),  whose  observations  were  made  chiefly  upon  B. 
hohneri^  agrees  with  Osterhout  as  regards  the  structure  of  the  carpo- 
gonium, but  in  the  spermatia  of  this  species  he  finds,  almost  invariably, 
two  nuclei.  Davis  ('96),  differing  from  both  Schmidle  and  Osteihout, 
claims  that  in  B.  moniliforme  Roth.,  B.  coerulescens  Sirodot,  and 
B.  boryanum,  the  trichogyne  is  a  distinct  cell  possessing  a  well  defined 
nucleus  and  chromophore,  and  connected  with  the  trichophore  by  a 
strand  of  protoplasm.  The  methods  used  by  Davis  at  the  time  were 
inadequate  for  the  better  differentiation  of  the  nucleus,  and  his  con- 
clusion is  in  all  probability  incorrect. 

The  copulation  of  the  spermatia  with  the  trichogyne  and  the  fusion 
of  the  sexual  nuclei  is  as  follows  :  One  to  several  spermatia,  which  are 
now  provided  with  a  cell-wall,  become  attached  to  the  trichogyne 
chiefly  near  the  end  (Fig.  45,  C,  and  Fig.  46,  D,  E).  After  the  disso- 
lution of  the  cell-membranes  at  the  point  of  contact  the  nucleus  of  the 
spermatium  enters  the  trichogyne  and  passes  down  through  it  into  the 
base  of  the  carpogonium.     The  canal  between  the  trichogyne  and  the 


ii8 


ASCOMYCETES  AND  RHODOPHYCE^. 


basal  part  of  the  carpogonium  now  becomes  narrower  and  is  finally 
closed  by  the  swelling  or  growth  of  the  cell-wall,  so  that  the  entrance 
of  other  male  nuclei  is  impossible  (Fig.  46,  D).  In  case  other  male 
nuclei  enter  the  trichogyne  from  other  adhering  spermatia,  as  frequently 
happens,  these  fragment  and  disappear,  and  the  same  fate  befalls  those 
nuclei  that  remain  in  other  adhering  spermatia.  Soon  after  the  male 
nucleus  enters  the  trichophore  it  fuses  completely  with  the  egg-nucleus 
(Fig.  46,  D,  E).     This  fact,  so  unmistakably  observed  by  Osterhout, 


Fig.  46. — Fusion  of  sexual  nuclei  and  immediate  subsequent  development  of  fecundated 
egg  in  Batrachospermuin. — (After  Osterhout.) 

D,  sexual  nuclei  in  act  of  fusing ;  an  empty  spermatium  adheres  to  tip  of  trichogyne. 

E,  later  stage ;  the  trichophore  has  increased  in  size  and  sent  out  a  protuberance ;  the 

empty  spermatium  which  has  copulated  with  the  trichogyne  has  furnished  the  male 
nucleus;  below  it  is  a  spermatium  with  a  nucleus,  and  above  to  the  left  is  one  in 
which  the  nucleus  has  undergone  fragmentation. 

leaves  no  room  for  doubting  the  existence  of  a  true  fecundation  in 
Batrachospermum . 

Schmidle  did  not  observe  the  actual  fusion  of  the  sexual  nuclei,  but 
he  concludes  that  the  same  takes  place.  He  asseits  that,  together  with 
the  two  nuclei  which  he  finds  in  the  spermatium,  a  portion  of  the  cyto- 
plasm also  enters  the  trichogyne,  while  the  plasma  membrane  remains 
behind,  save  in  exceptional  cases  in  which  the  spermatia  were  quite 
empty.  Davis  ('96)  having  failed  to  observe  the  entrance  of  the  male 
nucleus  into  the  egg-cell,  inclined  to  the  view  that  only  cytoplasmic 
contact  was  necessary  in   Batrachospermum  to  insure  the  further 


bUDRESNYA.  11^ 

development  of  the  spore  fruit  from  the  egg-cell.     Such  a  doctrine 
has,  of  course,  the  value  of  mere  conjecture  only. 

The  fusion  nucleus  increases  in  size  and  shows  clearly  a  single  large 
nucleolus  and  a  well-defined  threadwork  in  which  are  held  distinct 
chromatin  granules.  The  trichophore  now  begins  to  send  out  one  or 
more  protuberances  (Fig.  46,  E).  The  fusion-nucleus  divides,  and 
one  of  the  daughter-nuclei  passes  into  a  protuberance  which  is  then 
cut  off  by  a  transverse  wall.  By  a  repetition  of  this  process  many 
cells  are  produced,  each  containing  a  nucleus  which  is  a  descendant  of 
the  fusion-nucleus.  Each  of  the  cells  thus  borne  by  the  carpogonium 
will  give  rise  to  gonemoblast  filaments,  whose  end  cells  form  the 
carpospores. 

DUDRESNYA. 

From  the  foregoing  it  will  be  seen  that  the  sexual  process  and  the 
subsequent  development  of  the  fecundated  egg  in  Batrachospermum 
are  comparatively  simple,  but  in  the  vast  majority  of  the  Rhodophycece^ 
because  of  the  peculiar  structure  of  the  thallus,  the  details  in  these  pro- 
cesses are  extremely  difficult  to  follow  even  in  the  most  favorable  cases. 

In  the  better  known  representatives,  such  as  Dudresnya  and  the 
simpler  Callithamnion^  the  carpogonium  does  not  give  rise  to  the 
spore  fruit  (cystocarp),  as  in  Nemalion  (Wille)  and  Batrachosper- 
mum^  but  from  each  carpogonium  whose  egg-cell  has  been  fecundated 
a  number  of  filaments  (two  or  three  in  Dudresnya)  are  developed, 
which  fuse  with  certain  vegetative  cells,  and  from  which,  in  connection 
with  a  part  of  the  filament,  the  cystocarps  are  developed.  These  fila- 
ments are  the  ooblastema  Jilaments  of  Schmitz  ('83)  and  the  sporo- 
genous  filaments  of  Oltmanns  ('98).  The  vegetative  cells  with 
which  these  fuse  are  known  as  auxiliary  cells  or  brood  cells.  This 
fusion  of  the  sporogenous  filaments  with  auxiliary  or  brood  cells  was 
regarded  by  Schmitz  and  his  followers  as  a  second  fecundation,  a 
phenomenon  unparalleled  among  plants,  and  which,  as  Schmitz  put  it, 
was  contrary  to  all  tradition:  "  Einen  zweimaligen  Befruchtungsact 
im  Entwickelungskreise  einer  einzelnen  Species  anzunehmen,  dagegen 
straubt  sich  zur  zeit  die  botanische  Anschauung  vollstandiger,  das 
widerspricht  aller  Tradition." 

The  recent  researches  of  Oltmanns  ('9^  seem  to  show  what  is,  in 
all  probability,  the  true  significance  of  the  fusion  of  sporogenous  fila- 
ments and  auxiliary  cells.  He  maintains  that  the  fusion  of  the  sporoge- 
nous filament,  or  a  cell  of  the  same,  and  an  auxiliary  cell  is  not  a 
sexual  process,  since  it  is  only  a  cytoplasmic  and  not  a  nuclear  fusion 
that  takes  place.     Furthermore,  the  nuclei  of  the  carpospores,  as  in 


I20 


ASCOMYCKTES  AND  RHODOPHYCE^. 


Batrachospermum^  are  the  descendants  of  the  fusion  nucleus,  result- 
ing from  the  union  of  male  and  female  nuclei  in  the  carpogonium. 
The  nuclei  of  the  auxiliary  cells  never  take  a  morphological  part  in 
the  formation  of  the  carpospores.  According  to  this  view,  therefore, 
the  auxiliary  cells  are  merely  brood  cells,  their  fusion  with  the  cells 
of  the  sporogenous  filaments  representing  a  peculiar  condition  of  nutri- 


FiG.  47. — Carpogonium  and  its  development  subsequent  to  fecundation  in  Dudresnya 
purpurifera. — (After  Oltmanns.) 

A,  carpogoniai  branch  with  young  carpogone  at  upper  end. 

B,  later  stage  ;  nucleus  of  carpogonium  lies  some  distance  up  in  trichogyne,  whose  end  is  twisted. 

C,  still  later  stage ;  nucleus  lies  in  coiled  part  of  trichogyne. 

D,  carpogoniai  branch  after  fecundation;  fusion  of  sporogenous  filaments  {/"with  auxiliary  cells; 

az,  auxiliary  cells. 
K,  later  stage ;  sz,  sporogenous  cells  ;  az,  auxiliary  cells. 

tion.  A  further  discussion  of  this  phenomenon  is  reserved  for  a  later 
paragraph. 

In  order  to  comprehend  fully  the  statements  of  the  preceding  para- 
graphs, it  will  be  necessary  to  follow  somewhat  in  detail  the  process 
involved  in  one  of  the  forms  referred  to,  as,  for  example,  Dudresnya. 

In  Dudresnya  furpurif era ^  according  to  Oltmanns  ('98),  the  car- 


DUDRESNYA.  121 

pogonium  is  developed  from  the  end  cell  of  a  short  branch  (Fig.  47, 
A),  whose  remaining  cells  give  rise  to  numei'ous  side  branches  (Fig. 
47,  B).  The  end  cells  of  these  side  branches  may  become  auxiliary 
cells.  The  trichogyne  is  unusually  long,  showing  spiral-like  turns 
either  at  its  middle  or  nearer  the  base.  The  nucleus  lies  in  the  ventral 
part  of  the  young  carpogonium.  Later  it  passes  up  into  the  tricho- 
gyne, and  when  the  carpogonium  is  ready  for  fecundation,  the  nucleus 
is  to  be  found  in  the  coiled  region  of  the  trichogyne  (Fig.  47,  C).  The 
spermatium  applies  itself  to  the  tip  of  the  trichogyne,  which  projects 
slightly  beyond  the  general  sui'face  of  the  thallus.  The  cell-walls  at 
the  point  of  contact  are  dissolved,  the  sperm-nucleus  passes  down  into 
the  trichogyne  and  fuses  with  the  egg-nucleus  in  a  manner  described 
by  Wille  ('94)  for  Nemalion . 

Oltmanns  did  not  observe  the  actual  fusion  of  the  sexual  nuclei  in 
Dudresnya^  but  in  repeated  instances  two  nuclei  were  seen  lying 
tolerably  near  each  other  in  the  trichogyne,  and  at  a  later  stage  a 
single  nucleus  was  found  in  the  ventral  part  of  the  carpogonium, 
which  he  regarded  as  the  fusion-nucleus.  The  union  was  observed, 
however,  in  Dasya  elegans^  and  personally  Oltmanns  believes  the 
fusion  in  Dudresnya  to  be  too  probable  to  justify  an  exhaustive  study. 
It  may  be  remarked  that  in  general  this  is  b}'  no  means  a  safe  principle 
to  follow. 

After  fecundation  the  base  of  the  carpogonium  (or  shall  we  say  the 
fecundated  egg-cell)  segments  into  cells  which  increase  in  size  and 
begin  to  grow  into  sporogenou.s  filaments.  In  Dudresnya  purpu- 
rifera  two  or  three  pf  such  filaments  arise  from  the  carpogonium, 
one  on  either  side,  with  sometimes  a  third  between  them  (Fig.  47, 
D,  sf^.  The  sporogenous  filaments,  which  soon  become  segmented 
into  cells  by  transverse  walls,  grow  downward  among  the  lateral 
branches  of  the  carpogonial  branch  and  fuse  with  some  of  the  end 
cells  of  these  branches,  which  have  become  auxiliary  cells  (Fig.  47,  E, 
az) .  Certain  cells  of  the  carpogonial  branch  may  function  also  as 
auxiliary  cells.  The  auxiliary  cells  are  distinguished  by  their  form  and 
denser  protoplasmic  contents.  Usually  only  one  cell  of  a  sporogenous 
filament  unites  with  an  auxiliary  cell  or  cells. 

The  filaments  continue  their  growth  in  length,  fusing  with  other 
auxiliary  cells  which  may  be  borne  upon  other  and  widely  separated 
vegetative  branches  (Fig.  49).  The  fusion  of  any  auxiliary  cell  with 
that  of  a  sporogenous  filament  represents  only  a  cytoplasmic  fusion 
and  not  a  sexual  act.  This  process  with  the  immediate  subsequent 
changes  is  briefly  as  follows :  As  soon  as  the  cell-walls  at  the  point 


122 


ASCOMYCETES  AND  RHODOPHYCE^. 


of  contact  dissolve,  the  cytoplasm  of  the  two  cells  becomes  continuous. 
The  nuclei  show  no  tendency  even  to  approach  each  other,  but,  on 
the  contrary,  that  of  the  cell  of  the  sporogenous  filament  seems  to 
repel  the  nucleus  of  the  auxiliary  cell,  as  this  one  generally  retreats 
from  its  former  central  position  to  the  side  farthest  removed  from  the 
point  of  contact  of  the  two  cells  (Fig.  47,  E,  and  Fig.  48,  A).  That 
part,  or  half,  of  the  fusion  cell  which  corresponds  to  the  sporogenous 
filament  now  begins  to  send  out  a  protuberance  into  which  the  sporoge- 


^ar 


Fig.  48. — Copulation  of  sporogenous  filaments  with  auxiliary 
cells,  and  origin  of  a  cystocarp  in  D.  purpuri/era.—{,khtT 
Oltmanns.) 

A,  a  sporogenous  filament  has  fused  near  its  end  with  an  aux- 
iliary cell ;  sK,  sporogenous  nucleus,  az,  auxiliary  cell. 

B,  sporogenous  filament  after  copulating  with  an  auxiliary  cell 
has  continued  its  development ;  protuberance  containing 
sporogenous  nucleus,  sK,  will  probably  give  rise  to  a  cys- 
tocarp ;  aK,  nucleus  of  auxiliary  cell. 

C,  a  sporogenous  cell  has  been  cut  off  from  the  filament  oppo- 
site point  of  fusion  with  an  auxiliary  cell;  sK,  sporogenous 
nucleus  ;  aK,  nucleus  of  auxiliary  cell. 

D,  later  stage;  j/,  a  very  young  cystocarp. 

nous  nucleus  and  dense  cytoplasm  pass  (Fig.  47,  E,  sz).  In  the 
earlier  developmental  stages  following  fecundation  this  protuberance 
develops  an  additional  branch  of  the  sporogenous  filament  which  is 
to  seek  and  fuse  with  other  auxiliary  cells  (Fig.  48,  A,  B).  In  case 
of  the  development  of  a  cystocarp  from  the  fusion  cell,  the  protube- 
rance in  question,  after  the  division  of  its  nucleus,  will  be  cut  off  as  a 
rounded  cell  (Fig.  48,  C),  which  will  give  rise  ultimately  to  a  spore 
fruit. 

In  Dudresnya  ^urfurifera  the  nuclei  of  auxiliary  cells  which 
have  fused  with  cells  of  the  sporogenous  filaments  tend  to  diminish  in 
size  and  disappear,  while  in  D.  coccinea  the  nucleus  of  the  auxiliary 
cell  may  remain  normal  and  divide.  In  no  case,  however,  do  these 
auxiliary  nuclei  show  any  disposition  to  fuse  with  a  sporogenous 
nucleus. 


DUDRESNYA. 


123 

The  development  of  the  sporogenous  filaments,  their  fusion  with 
auxiliary  cells,  and  the  origin  of  cystocarps  from  the  fusion  cells  will 
he  more  readily  understood  from  the  diagram  in  Fig.  49.     At  a,  after 


!  i 

I 


i  • 
t  i 

i  \ 


Fig.  49. — Diagram  showing  origin  of  sporogenous  filaments  and  their  union  with  various  auxiliary  cells 
in  Dudresnyacoccinea  :  parts  drawn  by  means  of  short  dashes  indicate  trichogyne  and  sporogenous 
filaments,  while  the  dots  indicate  the  auxiliary  cells  ;  a,  b,  c,  d,  places  where  sporogenous  filaments 
have  united  with  auxiliary  cells. — (After  Oltmanns.) 

the  sporogenous  filament  had  fused  with  the  auxiliary  cell,  the  spo- 
rogenous nucleus  divided,  one  daughter-nucleus  remaining  in  the  fusion 
cell,  the  other  passing  into  the  end  of  the  filament  which  is  cut  off  by 


Ii4  ASCOMYCETES  AND  RHODOPHYCE^. 

a  transverse  wall.  This  end  cell  continues  the  development  of  the 
sporogenous  filament,  which  in  turn  may  fuse  with  other  auxiliary 
cells.  At  <5,  c,  d  the  sporogenous  parts  of  the  fusion  cells  have  given 
rise  to  branches  which  will  produce  either  sporogenous  filaments  or 
spore  fruits,  as  shown  in  Fig.  48,  C,  D. 

In  all  authentically  known  cases  among  the  Rhodophycece  the 
structure  of  the  female  sexual  organ,  the  carpogonium,  or  we  may  say 
the  oogonium,  and  the  process  of  fecundation  is  essentially  the  same, 
but  the  development  of  the  cystocarps  from  the  fecundated  egg  differs 
widely  in  detail  among  the  various  genera.*  So  far  as  is  known  the 
sporogenous  filaments  reach  their  highest  development  and  complexity 
in  Dudresnya^  in  which,  as  we  have  seen,  the  fusion  of  each  of  the 
sporogenous  filaments  takes  place  with  a  greater  number  of  widely 
separated  auxiliary  cells.  In  other  forms,  such  as  Callithamnion 
and  Dasya  (Oltmanns,  '98),  in  which  only  one  or  two  closely  situ- 
ated auxiliary  cells  take  part  in  the  formation  of  the  cystocarps,  the 
sporogenous  filaments  may  consist  of  only  a  few  cells  at  most.  In 
these  cases  we  can  scarcely  speak  of  sporogenous  filaments,  but  rather 
of  sporogenous  cells.* 

The  relation  which  an  auxiliary  and  a  sporogenous  cell  sustain  to 
each  other  is  somewhat  different  in  the  several  known  genera.  As 
already  stated  for  Dudresnya^  the  sporogenous  part  of  the  fusion 
cell  (Fig.  48,  B,  C,  D)  gives  rise  to  the  cystocarp,  while  in  Gloeosi- 
pkonia  capillaris  (Oltmanns,  '98)  the  sporogenous  cell,  after  the 
fusion  of  its  contents  with  the  auxiliary  cell,  may  take  no  further  part 
in  the  development.  Its  cytoplasm  and  nucleus  pass  into  the  auxiliary 
cell,  and  a  cell-wall  is  formed  separating  the  old  cavity  of  the  spo- 
rogenous cell  from  the  auxiliary  cell.  From  the  auxiliary  cell  the 
cystocarp  is  now  developed.  A  similar  process  takes  place  also  in 
Callithamnion  and  Dasya.  Although  the  behavior  of  the  two  cells 
in  the  last  two  genera  named  suggests  a  greater  similarity  to  a  real 
fecundation  than  in  Dudresnya,  yet  the  nuclei  of  the  two  cells  never 
fuse.  The  sporogenous  cell  merely  leaves  its  original  abiding  place 
to  take  possession  of  the  auxiliary  cell,  using  it  as  a  basis  from  which 
to  develop  the  spore  fruit ;  for  the  nuclei  of  the  auxiliary  cell  either 
disappear,  or,  if  they  persist,  take  no  part  in  spore-fruit  formation. 
The  nuclei  of  all  the  cells  of  the  spore  fruit  are  descendants  of  the 
sporogenous  nuclei,  and  are  therefore  sporophytic  nuclei,  while  those 
of  the  auxiliary  cells  are  gametophytic.     The  process  occurring  in 

'  In  addition  to  the  authors  mentioned  above  see  also  Philips,  '95,  '96,  '97,  '98.  Osterhout,  '961 
Hassenkamp,  '02. 

»  See  Oltmanns,  '98,  Taf.  vii.  Figs,  ii-ao. 


DUDRESNYA.  1^5 

Callithamnion ,,  Glceoslphonia^  Dasya  and  others  is  after  all  not  so 
extraordinary  as  it  may  at  first  appear,  since  the  superior  significance 
of  the  nucleus  in  all  constructive  metabolism  of  the  cell  has  been 
thoroughly  demonstrated. 

If  the  doctrine  of  Oltmanns  be  correct,  and  the  facts  seem  to  justify 
his  conclusion,  we  have  in  the  sporogenous  filaments  of  Dudresnya 
and  similar  genera  of  the  Rhodofhycece  a  sporophyte^  which,  for  the 
purpose  of  nutrition,  fuses  with  auxiliary  cells,  and,  because  of  the 
better  nutrition,  is  capable  of  producing  several  spore  fruits.  The 
auxiliary  cells  must,  therefore,  be  regarded  merely  as  special  brood 
cells,  their  fusion  with  the  cells  of  the  sporogenous  filaments  being 
homologous  with  the  fusion  of  vegetative  cells. 

As  regards  the  existence  of  an  alternation  of  generations  in  the 
Rhodophycece^  there  still  remains  the  question  upon  which  De  Bary 
laid  some  stress,  namely,  that  in  the  Rhodophycece^  as  well  as  in  the 
Ascomycetes,  there  is  no  rounding  up  or  separation  of  the  egg  as  an 
independent  cell  in  the  oogonium,  such  as  occurs,  for  example,  in 
Coleochcete^  in  the  Bryophyta  and  Pteridophyta.  In  the  second 
place  the  determination  of  the  number  of  chromosomes  in  these  gen- 
erations and  the  point  in  the  life-cycle  at  which  the  numerical  reduc- 
tion of  the  chromosomes  takes  place  are  factors,  which,  in  the  light  of 
important  existing  theories,  must  be  taken  into  consideration.  The 
first  of  these  questions  may  be  of  comparatively  little  importance,  but 
an  alternation  of  generations  in  the  Rhodophycece  will  probably  not 
be  unqualifiedly  accepted  by  some  botanists  until  the  question  of  the 
chromosomes  is  definitely  settled,  or  until  the  full  significance  of  the 
reduction  is  beyond  question. 

A  comparison  of  the  process  of  fecundation  and  the  immediate  sub- 
sequent development  in  certain  Ascomycetes  and  Floridecs  reveals 
several  striking  parallels,  or,  shall  we  say,  homologies.  In  the  first 
place  the  female  sexual  organ  in  both  groups  is  in  all  probability 
homologous.  The  carpogonium,  or  oogonium,  of  the  Floridece^  with 
its  large  receptive  part,  the  trichogyne,  may  be  compared  directly  with 
the  oogonium  of  the  Discomycetes^  e.  g.^  Pyronema,  and,  perhaps, 
with  the  carpogonium  of  the  lichen  Collema.  The  presence  or 
absence  of  a  trichogyne  is,  moreover,  of  secondary  importance,  as  this 
organ  is  purely  an  adaptation  to  peculiar  environmental  conditions. 

All  representatives  of  this  type  of  sexual  reproduction  agree  in  that 
the  egg  does  not,  by  self-plasmolysis,  separate  itself  as  an  individual 
from  the  oogonium.  Whether  the  gametes  be  uninucleate  or  multi- 
nucleate is  of  little  importance  as  viewed  from  a  phylogenetic  standpoint. 


126  ASCOMYCETES    AND    RIIODOPHYCE^. 

Lastly,  the  development  of  the  gonemoblast  filaments  in  such  forms 
as  Batrachospermum  and  Nemalion  is  certainly  paralleled  in  the 
ascogenous  hyphae  of  Erysiphe^  and  for  the  same  reason  we  may  look 
upon  the  ascogenous  hyphas  of  Pyronema  and  Ascobolus  as  homolo- 
gous with  the  sporogenous  filaments  of  Dasya  and  Dudresnya.  The 
ascogenous  hyphae  obtain  food  later  in  their  development  from  con- 
tiguous vegetative  cells  existing  chiefly  for  that  purpose.  In  this  case 
a  cytoplasmic  fusion  is  not  necessary  for  the  purpose  of  nutrition, 
although  it  may  possibly  occur,  but  in  the  RhodophycecB^  because  of 
their  aquatic  habit,  the  sporogenous  filaments  must  fuse  with  the  brood 
cells  in  order  to  obtain  nourishment  from  them  in  the  most  effective  way. 

This  view  of  phylogenetic  relationship  is  made  more  probable  by 
the  researches  of  Thaxter  on  the  Laboulbeniacece ^  in  which  certain 
representatives  are  shown  to  be  transitional  between  the  Floridece  and 
the  Ascomycetes.  It  is  certain  that  the  Ascomycetes  resemble  the  red 
algse  more  than  they  do  the  lower  fungi,  yet,  as  we  may  conclude  with 
Harper,  "  whether  these  resemblances  are  the  result  of  blood  relation- 
ship or  merely  due  to  that  similarity  in  the  chemical  constitution  of 
the  protoplasm  of  different  organisms,  which  under  similar  conditions 
enables  it  to  develop  structures  nearly  related  in  appearance  out  of 
rudiments  which  may  be  extremely  diverse,  is  likely  to  remain  a 
puzzling  question." 

COLLEMA. 

The  much  discredited  doctrine  of  Stahl  ('77)  and  others  concerning 
sexuality  in  certain  lichens  has  received  fresh  confirmation  recently  by 
the  researches  of  Baur  ('98)  and  Darbyshire  ('99).  Although  neither 
cell  nor  nuclear  fusion  has  been  established  beyond  all  doubt,  yet  the 
morphological  value  of  the  sexual  organs  can  not  be  very  well  ques- 
tioned. 

According  to  Stahl,  as  is  well  known,  the  sexual  organs  of  Collema 
microphyllutn  occur  in  large  numbers  especially  upon  the  illuminated 
edges  of  the  rapidly  growing  vegetative  lobes  of  the  thallus.  The 
carpogonium  arises  some  little  distance  beneath  the  upper  surface  of 
the  thallus  as  an  ordinary  hyphal  branch.  The  lower  part,  the  asco- 
gonium,  consists  of  a  row  of  short  cells  coiled  up  somewhat  in  the 
form  of  a  corkscrew,  which  are  distinguished  from  the  other  hyphal 
cells  by  their  larger  diameter  and  denser  plasmic  contents  (Fig.  50,  A). 
The  number  of  cells  composing  the  ascogonium,  which  makes  two  or 
three  turns,  varies  considerably,  but  may  often  reach  twelve.  The 
ascogonium  is  continued  into  a  straight  filament,  the  trichogyne,  which 
extends  to  the  upper  surface  of  the  thallus.     The  cells  of  the  tricho- 


COI.I.EMA. 


127 


gyne  are  smaller  in  diameter  than  those  of  the  ascogonium,  and  their 
number  varies  in  the  species  examined  from  six  to  twenty-four.  A 
sharp  demarkation  between  trichogyne  and  ascogonium  does  not  exist. 

The  end  of  the  trichogyne  which 
projects  above  the  surface  of  the 
thallus  is  generally  short  and  cylin- 
drical or  flask-shaped.  In  rare  cases 
it  ends  in  two  short  and  nearly  equal 
branches.  The  free  surface  of  this 
end  cell  is  covered  by  a  viscid  sub- 
stance which  facilitates  the  adherence 
of  the  spermatia  that  escape  in  large 
numbers  during  moist  weather  from 
the  flask-shaped  male  organs,  the 
spermagonia. 

Baur  ('98),  who  studied  Collema 
crispum,  confirms  Stahl's  observa- 
tions, and  gives  additional  informa- 
tion concerning  details  of  cell  struc- 
ture. The  terminal  cell  of  the  trich- 
ogyne in  Collema  crispum^  which 
projects  above  the  surface  of  the 
thallus,  is  much  larger  than  the  other 
cells  of  this  organ,  being  longer, 
somewhat  swollen  at  the  middle,  and 
terminating  in  a  point  (Fig.  50,  B). 
It  is  also  provided  with  a  viscid 
coating. 

Each  cell  of  the  entire  carpogo- 
nium  possesses  a  nucleus  of  the  typi- 
cal structure.  The  transverse  walls 
between  the  cells  are  not  broken 
down,  though  each  reveals  a  small 
pit,  such  as  is  present  in  the  trans- 
verse septa  of  vegetative  hyphae. 
In  four  cases  Baur  found  empty 
spermatia  attached  to  the  end  of  the  trichogyne,  whose  cells  showed 
the  same  signs  of  degeneration  described  by  Stahl.  The  cells  in  the 
upper  part  were  collapsed,  the  cross-walls  much  swollen,  and  no  nuclei 
could  be  seen  in  them.  The  septa  between  the  lower  cells  of  the 
trichogyne  were  clearly  broken  down.  Each  cell  of  the  ascogonium 
contains  at  first  one  nucleus,  and  since  each  gives  rise  to  ascogenous 


Fig.  so. — Carpogonium  of  Collema  crispum. 
(After  Baur.) 

A,  mature  carpogonium ;  trichogyne  ends  in 

large  receptive  cell  which  projects  above 
surface  of  thallus. 

B,  receptive  cell  with  which  a  spermatium 

has  fused. 


128  ASCOMVCETES  AND  RHODOPHYCE^. 

hyphae,  the  pores  in  the  septa  may  be  associated  with  some  part  of  the 
process  of  fecundation.  Baur  is  inclined  to  regard  the  first  cell  of  the 
ascogonium  as  the  egg-cell,  attributing  to  the  rest  the  role  of  auxiliary 
cells  similar  to  that  described  by  Oltmanns  for  certain  Ploridece. 

In  many  cases  carpogonia  were  found  which  showed  no  evidence  of 
development  into  apothecia,  their  cells  giving  rise  merely  to  vegetative 
hyphae.  In  these  cases  no  spermatia  were  found  attached  to  the 
receptive  cells  of  the  trichogyne. 

The  discovery  of  a  carpogonium  in  Physcia  fulverulenta  (Schreb.) 
Nyl.  by  Stahl  and  Lindau  has  been  confirmed  by  Darbyshire.  He 
finds,  however,  that  the  cells  of  the  carpogonium  become  connected  by 
broad  strands  of  protoplasm  so  as  to  form  almost  a  single  multinu- 
cleated cell.  Darbyshire  shows  also  the  falsity  of  Lindau's  view, 
namely,  that  the  trichogyne  is  merely  a  boring  hypha  which  serves  to 
break  a  way  upward  through  the  thallus  for  the  apothecium. 

From  the  investigations  of  the  authors  mentioned  there  seems  to  be 
no  doubt  that,  in  the  genera  in  question,  the  development  of  the  spore 
fruit  is  the  result  of  a  true  sexual  process. 


CHAPTER  VI.— ARCHEGONIATES. 

The  preceding  chapters  have  been  devoted  to  the  process  of  fecun- 
dation in  various  typical  and  well  known  Thallophyta^  with  the 
exception  of  the  Characecs^  if  we  may  speak  of  this  group  as  belonging 
properly  to  the  Thallophyta.  Owing  to  the  closer  resemblance  of 
both  sexual  organs  and  gametes  to  those  of  certain  Archegoniates^  it 
has  been  deemed  best  to  refer  to  the  Ckaracece  in  connection  with 
those  plants. 

Because  of  our  meager  knowledge  of  the  development  of  the  sperma- 
tozoids,  and  the  union  of  the  sexual  nuclei  in  liverworts  and  mosses,  I 
have  omitted  a  discussion  of  the  process  in  these  groups  and  have  dealt 
more  fully  with  sexual  reproduction  in  certain  Pteridophyta  and 
gymnosperms. 

The  discovery  of  spermatozoids  in  Cycas  by  Ikeno  and  Hirase,  and 
in  Zamia  by  Webber,  and  a  more  accurate  knowledge  of  the  develop- 
ment of  these  structures  in  the  Pteridophyta  have  aroused  an  unusually 
keen  interest  in  the  study  of  the  sexual  cells  and  the  phenomena 
accompanying  their  union  both  in  these  and  in  the  higher  plants.  In 
presenting  the  phenomena  relating  to  the  sexual  process  in  the  Arche- 
goniates^  we  shall  confine  ourselves  largely  to  Onoclea  and  Gymno- 
gramme  among  the  Pteridophytes  and  to  Cycas^  Zamia^  Ginkgo^ 
and  Pinus  of  the  gymnosperms ;  for  it  is  in  certain  species  of  these 
genera  that  the  process,  in  so  far  as  it  has  been  followed  with  the  use 
of  later  methods  of  research,  is  best  known. 

PTERIDOPHYTA. 

Until  recently  the  spermatozoid  of  the  Pteridophyta  was  generally 
conceded  by  many  of  the  most  competent  investigators  to  consist 
merely  of  a  transformed  nucleus  with  cilia  of  an  obscure  cytoplasmic 
origin.  This  view  was  due  very  largely  to  the  methods  of  fixing  and 
staining  used,  which,  as  we  now  know,  were  inadequate  to  bring  out 
with  definite  clearness  the  more  delicate  cytoplasmic  structures  of  the 
cell. 

In  recent  years  Belajeff,  Shaw,  and  others  have  applied  improved 
cytological  methods  to  the  study  of  the  development  of  the  sperma- 
tozoid in  Gymnogramme^  Onoclea^  Marsilia  and  Equisetum.  In 
certain  species  of  these  genera,  they  have  found  that  the  mature 
spermatozoid  consists  of  a  nucleus  and  a  delicate  band  or  wing  of 


13© 


ARCIIEGONIATES. 


cytoplasm  along  whose  outer  edge  is  a  delicate  thread  or  band  derived 
also  from  the  c)i;oplasm,  and  from  which  the  cilia  are  developed  (Fig. 
52,  A).  Belajeff  was  the  first  to  call  attention  to  the  cilia-bearing 
band,  which  he  observed  in  the  development  of  the  spermatozoid  in  a 
fern  and  in  Equisetum.  He  also  reported  a  similar  body  in  Chara. 
In  speaking  of  the  body  which  gives  rise  to  the  cilia-bearing  band, 
Belajeff  used  the  term  "  Nebenkern,"  because  of  its  apparent  resem- 
blance to  a  body  of  that  nature  in  the  spermatid  of  certain  animals. 
In  1897  Webber  described  the  development  of  the  cilia-bearer  in  the 
spermatozoid  mother-cell  of  Zatnia^  and  gave  to  it  the  name  bleph- 


G  H 

Fig.  5f. — Development  of  sperma- 
tozoid in  GytHtiogratnme  sul- 
fhurea,—{A.(\.CT  Belajeff.) 


A,  grandmother-cell  of  spermatozoid  with  two  primordia  of  bleph- 
aroplasts. 

B,  two  spermatozoid  mother-cells,  each  with  its  blepharoplast 
primordium. 

C,  spermatozoid  mother-cell  rounded  off. 

D,  the  young  blepharoplast  has  begun  to  elongate. 

E,  stage  a  little  older  than  D. 

F,  blepharoplast  much  elongated  ;  its  anterior  end  extends  out  to 

plasma  m<jmbrane. 

G,  transformation  of  nucleus  has  begun  ;  it  is  somewhat  pear- 
shaped,  being  concave  on  side  turned  from  blepharoplast;  end 
which  will  be  anterior  in  mature  sperm  is  pointed. 

H,  later  stage ;  cilia  have  been  developed  from  the  blepharoplast. 


aroplast.^  Ikeno  and  Hirase,  who  were  the  first  to  discover  the 
.spermatozoid  in  certain  gymnosperms,  described  the  development  of 
the  cilia-bearing  band  in  the  spermatozoid  of  Cycas  and  Ginkgo. 

Belajeff  and  the  two  Japanese  investigators  consider  the  body 
developing  into  the  blepharoplast  as  a  centrosome.  The  author  is 
convinced  that  it  has  been  clearly  proved  that  the  blepharoplast  is  not 
a  centrosome,  nor,  as  yet,  has  any  phylogenetic  relationship  been 
shown  to  exist  between  the  blepharoplast  and  the  centrosome  as  we 
know  this  structure  in  plants.'^ 

THE  SPERMATOZOID. 

The  development  of  the  spermatozoid  in  Onoclea,  as  described  by 
Shaw  ('98),  is  quite  similar  to  that  of  Gymnogramme  according  to 

'  From  ^\«ij>apis,  eyelash  or  cilium  ;  and  irAacTTOS,  formed. 
'  See  Introduction,  p.  46. 


PTERIDOPHYTA. 


131 


Belajeff  ('98).  Prior  to  the  division  of  the  grandmother-cell  of  the 
spermatozoid,  /.  e.,  the  last  cell-division  in  the  spermogenous  tissue 
of  the  antheridium,  which  gives  rise  to  the  cells  that  develop  directly 
into  the  spermatozoids,  there  appears  on  opposite  sides  of  the  nucleus 
a  small  globular  body  of  a  homogeneous  structure,  staining  rather 
densely  (Fig.  51,  A).  These  bodies  are  not  provided  with  any  radia- 
tions. In  Onoclea  there  is,  immediately  surrounding  the  nucleus,  a 
region  of  less  granular  cytoplasm  from  which,  undoubtedly,  the  weft 
of  spindle  fibers  is  developed.  These  bodies,  which  are  the  primordia 
of  the  blepharoplasts,  lie  just  at  the  outer  edge  of  this  region  or  weft 
(Fig.  51,  A).  In  the  telophase  a  blepharoplast  primordium  lies  near 
the  depression  of  each  daughter-nucleus,  very  near  the  pole  of  the 
spindle  (Fig.  51,  B,  C).  Each  appears  now  to  be  a  hollow  globular 
vesicle.  Soon  after  cell-division  is  completed  the  development  of  the 
daughter-cells  directly  into  spermatozoids  begins.  The  blepharoplast 
primordium  becomes  somewhat  lens-  or  crescent-shape  in  Gymno- 
grainme,,  with  the  concave  side  turned  toward  the  nucleus.  The 
nucleus  at  the  same  time  becomes  flattened  upon  one  side  and  gradu- 
ally passes  into  a  crescent-  or  pear-shaped  body  (Fig.  51,  D,  E).  The 
blepharoplast  has  elongated  into  a  thread  or  band,  which  follows  the 
convex  side  of  the  nucleus  and  is  rather  close  to  it.  One  end  of  the  band 
now  extends  beyond  that  end  of  the  nucleus  which  will  be  anterior  in 
the  mature  spermatozoid  (Fig.  51,  F,  G).  With  further  development 
the  blepharoplast  moves  away  from  the  nucleus  to  a  position  just 
beneath  the  plasma  membrane  (Fig.  51,  H).  At  this  stage  the  cyto- 
plasm in  Onoclea  (Shaw,  '98)  shows  a  depression  corresponding  to  the 
concave  side  of  the  nucleus.  At  about  this  period  in  the  development 
in  Gymnogramme^  according  to  Belajeff,  the  cilia  make  their  appear- 
ance as  outgrowths  of  the  blepharoplast.  The  nucleus  elongates, 
becoming  more  slender,  and  gradually  assuming  a  spiral  or  corkscrew 
shape  of  two  or  three  turns.  In  the  mature  spermatozoid  (Fig.  52, 
A)  the  nucleus  is  thicker,  tapering  abruptly,  and  sometimes  to  a  point, 
at  the  posterior  end,  but  gradually  forward  into  a  slender  anterior  end. 
It  is  oval  in  cross  section,  or,  in  sonie  cases,  slightly  flattened  on  the 
inner  side,  especially  in  the  thicker  posterior  part.  In  mature  sperma- 
tozoids of  Onoclea  struthiopteris^  fixed  and  stained  on  the  slide,  the 
cytoplasmic  part  seems  to  be  in  the  form  of  a  band  which  conforms  to 
the  spiral  course  of  the  nucleus.  It  is  broadest  at  the  anterior  end, 
which  extends  a  short  distance,  about  one  or  two  turns,  beyond  the 
anterior  end  of  the  nucleus,  but  it  narrows  gradually  backward,  dis- 
appearing at  a  point  which  marks  the  thickest  part  of  the  nucleus 


132 


ARCHEGOXIATES. 


(Fig.  52,  A).  Along  the  outer  edge  of  the  cytoplasmic  band  extends 
the  blepharoplast  as  a  thread  or  narrow  band  from  which  the  cilia  arise. 
The  blepharoplast  reaches  almost  or  quite  to  the  anterior  extremity  of 
the  cytoplasmic  part,  but  it  cannot  be  traced  farther  back  than  the 
posterior  extremity  of  the  cytoplasmic  part,  although  it  may  extend 
some  distance  farther  as  a  delicate  thread  closely  applied  to  the  nucleus. 
The  blepharoplast  is  broadest  at  its  anterior  end,  where  it  seems  to  be 
not  perfectly  flat,  but  curved,  appearing  as  a  double  line,  or  in  cross 
section  as  a  shallow  U.  It  is,  however,  very  small,  so  that  the  exact 
shape  is  difficult  to  determine  with  certainty.  As  already  stated,  it 
becomes  a  very  delicate  thread  at  the  posterior  end  which  is  brought 


Fig.  52. — Two  mature  spermatozoids  drawn  from  specimens  that  were  fixed  and  stained 

upon  the  slide  a  few  minutes  after  their  escape  from  the  antheridium. 

A,  OnocUa  strutkiopteris  ;  B,  Marsilia  vestita. 

close  to  the  nucleus  by  the  narrowing  of  the  cytoplasmic  band.  It  is 
probably  for  this  reason  that  it  cannot  be  traced  after  coming  into  con- 
tact with  the  nucleus.  There  is  nothing  to  indicate  that  the  blepharo- 
plast extends  to  the  posterior  end  of  the  nucleus.  The  cilia  begin 
at  a  short  distance  from  the  anterior  end,  and  extend  backward  about 
two  and  one-half  or  three  turns.  Their  length  equals  or  even  exceeds 
that  of  the  spermatozoid  when  extended. 

Judging  from  Belajeff's  figure  of  a  mature  spermatozoid,  it  would 
seem  that  the  cytoplasm  envelops  the  entire  nuclear  portion,  but  in  my 
own  preparations,  which  were  made  by  killing  and  staining  the  sper- 
matozoids upon  the  slide  after  they  had  escaped  from  the  antheridium, 
no  cytoplasmic  mantle  was  seen  to  surround  the  posterior  part  of  the 
nucleus.  Thom  ('99)  states  also  that  the  whole  nucleus  is  surrounded 
by  a  cytoplasmic  envelope.  It  is  possible,  of  course,  that  the  plasma 
membrane,  or  even  a  thin  layer  of  cytoplasm,  may  envelop  the  nuclear 
portion.     The  nucleus  usually  appears  homogeneous  in  structure,  but 


PTERIDOPHYTA. 


133 


in  some  cases  in  which  the  stain  was  well  washed  out  the  structure 
appeared  coarsely  reticulate  or  granular.  This  was  observed  in  sper- 
matozoids  of  Onoclea  struthiopteris  that  were  killed  on  the  slide  in 
chrom-osmic-acetic  acid  and  stained  in  safranin  gentian-violet  and 
orange  G. 

The  posterior  turns  of  the  spermatozoid  embrace  the  vesicle,  which 
presents  a  very  fine  reticulum,  and  in  which  coarse  granules  are  held, 
among  them  being  small  starch  grains.  The  author  has  observed  that 
the  vesicle  of  Onoclea  struthiopteris  became  separated  from  the 
spermatozoids  a  short  time  after  their  escape  from  the  antheridium ; 
for,  of  the  many  hundreds  fixed  and  stained  upon  the  slide  a  few 
minutes  after  their  escape  from  the  antheridia,  relatively  few  were 
found  with  the  vesicle  adhering. 

The  development  of  the  spermatozoid  of  Marsilia^  according  to 
Shaw  ('98)  and  Belajeff  ('99),  differs  in  certain  important  details 
from  that  of  Onoclea.  As  this  process  is  known  in  so  few  of  the 
Pteridophyta^  it  is  perhaps  well  to  present  briefly  the  facts  as  they 
are  known  in  one  of  the  heterosporous  forms. 

At  the  close  of  the  second  from  the  last  division  in  the  spermogenous 
tissue  of  Marsilia  vestita,  or  that  leading  to  the  great-grandmother- 
cell  of  the  spermatozoid  (the  primary  spermatocyte  of  Shaw),  there 
appears  at  each  pole  of  the  spindle,  or  near  it  close  to  the  daughter- 
nucleus,  a  small  body  which  is  called  by  Shaw  a  blepharoplastoid . 
During  the  resting  stage  of  the  nucleus  the  blepharoplastoid  seems  to 
divide.  The  two  halves  increase  in  size  and  remain  together  near  the 
nucleus.  As  soon  as  the  nucleus  of  the  great-grandmother-cell  begins 
to  divide,  the  pair  of  blepharoplastoids  move  away  from  the  nucleus 
and  remain  at  a  position  in  the  cytoplasm  between  one  pole  of  the 
spindle  and  the  equatorial  plane,  until  the  metaphase,  or  early  anaphase, 
when  they  disappear.  About  the  same  time,  or  a  little  later,  a  small 
blepharoplast  appears  near  each  pole  of  the  spindle.  At  the  close  of 
the  division  the  blepharoplast  lies  near  the  nucleus  of  the  grand- 
mother-cell of  the  spermatozoid  (secondary  spermatocyte  or  sperma- 
tocyte mother-cell  of  Shaw).  It  now  divides,  and  the  two  daughter 
blepharoplasts  increase  in  size  and  separate  from  each  other,  at  the 
same  time  moving  away  from  the  nucleus  (Fig.  53,  A,  B).  Each 
takes  a  position  near  the  pole  of  the  future  spindle  but  always  a  little 
to  one  side  of  its  longitudinal  axis.  They  increase  in  size  and  remain 
apparently  unchanged  in  structure  until  the  anaphase,  when  each  seems 
to  be  hollow  (Fig.  53,  B,  C). 

As  soon  as  the  nucleus  of  the  spermatozoid  mother-cell  (spermatid) 


134 


ARCHEGONIATES. 


is  formed,  a  small  eccentric  body  appears  in  each  blepharoplast  (Fig. 
53,  D),  then  several,  so  that  it  appears  as  if  the  blepharoplast  had 
broken  up  into  a  group  of  small  bodies  (Fig.  54,  E).  Out  of  these 
bodies  is  developed  the  band,  which  elongates,  and  together  with  the 
nucleus  moves  toward  the  plasma  membrane  of  the  cell  (Fig.  54,  F,  G). 
In  cross  section  the  band  is  broadly  U-shaped,  but  when  seen  from 
above  it  appears  as  a  double  line  (Fig.  54,  H).  The  band  continues 
to  elongate  until  finally  a  spiral  is  formed,  which  makes  five  or  more 
turns  about  the  hemispherical  half  of  the  cell  (Fig.  54,  I).  The 
nucleus  also  elongates,  becoming  sausage-shaped,  and  lies  in  close 
contact  with  the  larger  turns  of  the  blepharoplast.  The  mature  sper- 
niatozoid  in   Marsilia   is  composed,   therefore,    of   a  blepharoplast, 


Fig.  53. — Blepharoplast  primordium  during  division  of  grandmother-cell  of  spermatozoid  in 
Marsilia,  vestita. — (After  Shaw.) 

A,  the  two  primordia  of  the  blepharoplasts  lie  in  cytoplasm  some  distance  from  nucleus. 

B,  they  are  now  on  opposite  sides  of  the  nucleus  but  a  little  to  one  side  of  median  line. 

C,  the  nucleus  is  iu  spindle  stage  of  division;  the  young  blepharoplasts  lie  near  the  respective  poles  of 

spindle. 

D,  telophase  of  division;  blepharoplast  rudiment  at  pole  of  each  nucleus  contains  a  dense  granule. 

consisting  of  a  funnel-shaped  spiral  of  about  ten  or  more  turns,  and  a 
sausage- shaped  nucleus  without  a  definite  visible  structure,  which  is 
connected  with  the  three  larger  posterior  turns  of  the  blepharoplast 
(Fig.  52,  B).  The  posterior  end  of  the  blepharoplast,  which  is  usually 
bent  in  the  shape  of  a  hook,  extends  beyond  the  nucleus.  The  rela- 
tively large  vesicle  is  embraced  by  the  larger  posterior  tui^ns  of  the 
blepharoplast.  In  Marsilia  vestita  the  author  observed  that  the 
vesicle  remains  adhering  to  the  spermatozoid  for  a  longer  time  than  in 
Onoclea  struthiopteris.  The  vesicle  consists  of  a  delicate  cytoplasmic 
reticulum,  in  which  are  held  large  starch  and  protein  granules.  The 
numerous  cilia  (the  spermatozoids  were  fixed  and  stained  upon  the 
slide)  spring  from  the  middle  and  posterior  coils,  the  two  or  three 
anterior  coils  being  free  from  them.  In  some  cases  observed  the  cilia 
extended  almost  to  the  posterior  end  of  the  blepharoplast.  As  soon 
as  the  vesicle  drops  off,  the  spermatozoid  becomes  much  elongated, 
losing  its  pronounced  funnel-shape. 


PTKRIDOPHYTA. 


135 


Belajeff  ('99) ,  who  has  also  studied  the  development  of  the  sperma- 
tozoid  in  Marsilia^  agrees  with  Shaw  in  so  far  as  the  transformation 
of  the  primordium  of  the  blepharoplast  into  the  mature  cilia-bearing 
organ  is  concerned,  but,  as  regards  the  earlier  behavior  of  the  primordia, 
these  observers  disagree  in  certain  important  particulars.  Belajeff, 
who  regards  the  blepharoplast  as  a  centrosome,  finds  that  in  the 
division  of  the  grandmother-cell  of  the  spermatozoid,  the  primordia, 
which  lie  some  distance  from  the  nucleus,  divide,  and  a  faint  central 
spindle  is  formed  between  the  daughter  primordia.  This  structure,  he 
mamtains,  gives  rise  to  the  karyokinetic  spindle  just  as  in  some  animal 


Fig.   54. — Transformation   of  mother-cell   into  mature  spermatozoid   in 
Mars  ilia  vestita. — (After  Shaw.) 

E,  two  spermatozoid  mother-cells ;  each  rudiment  of  blepharoplast  has 
become  a  group  of  granules. 

F,  spermatozoid  mother-cell ;  the  blepharoplast  {b)  is  much  elongated. 
c,  cytoplasm  ;  s,  starch  grains. 

G,  the  thread-like  blepharoplast  and  bean-shaped  nucleus  lie  dose  to 
plasma  membrane. 

H,  an  older  suge  seen  from  above;  it  is  app.irent  that  blepharoplast  is  a 
band  concave  on  the  outside. 
'  I,  the  blepaaroplast  and  sausage-shaped  nucleus  {,k)  make  several  spiral 

turns  within  the  cell  close  to  plasma  membrane. 

cells,  and  concludes,  therefore,  that  the  blepharoplast  primordia  are 
centrosomes.  The  author  has  already  dealt  with  this  matter  in  the 
introductory  chapter,  and  a  further  discussion  will  not  be  given  here. 

In  Equisetum  Belajeff  has  found  that  the  spermatozoid  develops  in 
a  manner  similar  to  that  of  the  fern,  and  there  are  good  reasons  for 
believing  that  the  process  of  development  is  much  the  same  in  the 
majority  of  archegoniates,  although  our  knowledge  is  yet  too  meager 
to  warrant  any  sweeping  generalization. 

It  seems  fitting  in  this  connection  to  compare  the  mature  spermato- 
zoid of  the  Characece  with  that  of  the  fern.  Belajeff  ('94)  has  shown 
that  in  the  development  of  the  spermatozoid  of  Charafcetida  the  two 
cilia  are  borne  by  a  thread-like  body  which  arises  in  the  cytoplasm  in 
a  manner  similar  to  the  blepharoplast  of  the  fern.     The  spermatozoid, 


136  ARCHKGONIATES. 

as  in  the  Pteridophyta  and  gymnosperms^  is  a  transformation  of  the 
entire  contents  of  the  cell,  and  we  may  with  much  propriety  regard  the 
spermatozoid  of  Chara  and  that  of  the  fern  as  homologous  structures. 
But  whether  we  are  dealing  with  real  homologies,  or  only  with  striking 
analogies,  is  certainly  a  question  concerning  which  there  may  be  some 
diversity  of  opinion. 

The  fate  of  the  spermatozoid  of  Chara  after  penetrating  the  q%%  and 
the  union  of  the  two  sexual  nuclei  is  practically  unknown  in  detail,  and 
a  further  discussion  of  the  process  of  fecundation  in  the  absence  of  more 
facts  would  seem  without  value,  since  it  is  not  the  purpose  to  enter  here 
into  any  discussion  of  the  homologies  of  the  sexual  organs  of  the 
Characeae  with  those  of  the  Archegoniates. 

THE  EGG-CELL  AND  FECUNDATION. 

In  more  recent  years  the  process  of  fecundation  has  been  observed 
in  various  genera  of  the  Felicinecs  by  Campbell,  in  Onoclea  by  Shaw, 
and  in  Adianium  and  Aspidium  by  Thom.  The  author  has  followed 
the  process  in  Onoclea  struthlopteris,  and  his  observations  confirm 
those  of  Shaw,  who  has  traced  the  behavior  of  the  sexual  nuclei  in 
great  detail  in  Onoclea  sensibilis. 

Soon  after  the  division  which  cuts  off  the  ventral  canal-cell,  and 
before  the  archegonium  of  Onoclea  struthiopteris  is  full  grown,  the 
three  central  cells  contain  fine-meshed  and  densely  granular  cytoplasm. 
Their  nuclei  are  in  the  resting  stage.  The  wall  between  egg  and 
ventral  canal-cell  is  generally  arched  slightly  downward  into  the  egg- 
cell.  This  wall  is  laid  down  in  this  position,  at  least  in  many  cases, 
and  the  concave  upper  surface  of  the  ^^<g  does  not  seem  to  be  due  to 
pressure  from  the  ventral  or  neck  canal-cell. 

As  the  archegonium  matures  it  increases  in  size,  and  the  cytoplasm 
of  the  central  cells  becomes  looser.  A  rather  large  vacuole  has  been 
observed  in  the  ventral  canal-cell  in  the  mature  organ.  It  is  well 
known  that  in  Onoclea  the  nucleus  of  the  neck  canal-cell  often  divides, 
but  a  division  of  the  cell  does  not  follow,  except,  possibly,  in  rare 
cases.  The  daughter-nuclei  are  reconstructed  and  lie  usually  close  to 
each  other.  The  author  has  observed  in  several  instances  that  the 
division  of  the  neck-canal  nucleus  took  place  at  exactly  the  same  time 
as  the  division  of  the  central  cell  which  cuts  off  the  ventral  canal-cell. 
Whether  any  special  significance  should  be  attached  to  this  phenome- 
non the  author  is  unable  to  state.  Observers  have  often  been  tempted 
to  consider  the  ventral  canal-cell  as  a  rudimentary  ^g^^  but  if  there  be 
good  grounds  for  such  a  view  it  is,  perhaps,  as  much  in  harmony 
with  the  facts  to  regard  the  neck  canal-cell  or  cells  as  aborted  eggs. 


PTERIDOPHYTA. 


^37 


The  entrance  of  the  living  spermatozoid  into  the  neck  of  the  arche- 
gonium  and  its  passage  down  to  the  egg  is  easily  followed.  In  fact, 
the  phenomenon  is  a  matter  of  common  observation  in  elementary 
classes.  It  is  only  necessary  to  mount  prothallia  with  mature  arche- 
gonia  ventral  side  up  in  a  drop  of  water,  to  which  are  added  several 
clean  male  prothallia  that  contain  ripe  antheridia,  and  which  have  been 
kept  in  dry  air  for  a  short  time  previous  to  the  operation.  The  ripe 
archegonia  will  open,  and  in  a  few  minutes  numerous  spermatozoids 
which  have  escaped  on  being  placed  in  the  water  will  be  found  swim- 
ming about  the  opening  of  the  archegonium,  having  been  attracted 
thither  by  the  extruded  substance.  Many  enter  the  neck,  and  several 
may  reach  the  egg-cell.  The  author  has  observed  instances  in  which 
the  number  of  spermatozoids  endeavoring  to  enter  the  archegonium 
was  so  great  that  they  formed  a  plug  which  almost  completely  closed 
the  opening  in  the  neck. 

Since  the  interesting  researches  of  Pfeffer  ('84)  it  has  been  known 
that  the  mucilaginous  substance  formed  from  the  neck-canal  and 
ventral-canal  cells  acts  as  a  chemotactic  stimulus  upon  the  spermato- 
zoids. Pfeffer  found  that  the  spermatozoids  of  ferns  are  attracted  by 
malic  acid  and  its  salts  in  very  dilute  solutions.  A  solution  of  o.ooi  grm. 
per  cent,  is  sufficient  to  bring  about  a  positive  chemotactic  reaction. 

Buller  (1900)  found  that  in  addition  to  malic  acid  and  its  salts,  many 
organic  and  inorganic  salts,  widely  occurring  in  the  cells  of  plants, 
exercise  a  positive  chemotactic  stimulus  upon  the  spermatozoids  of 
certain  ferns.  Among  the  organic  salts  which  were  found  to  attract 
are  tartrates,  potassium  oxalate,  potassium  acetate  and  sodium  formate. 
Among  the  inorganic  salts  are  phosphates,  sulphates,  potassium  nitrate 
and  potassium  chloride.  Organic  substances  which  were  found  to  act 
indifferently  are  grape  sugar,  cane  sugar,  lactose,  amylodextrine, 
glycerine,  alcohol,  asparagin  and  ui-ea.  "Inorganic  salts  not  appre- 
ciably attracting  are  the  chlorides  and  nitrates  of  sodium,  ammonium 
and  calcium,  and  also  lithium  nitrate.  Of  the  four  free  acids  which 
seem  to  be  most  widely  found  in  cell-sap,  namely,  malic,  oxalic,  tartaric 
and  citric,  only  malic  acid  attracts."  The  concentration  of  malic  acid 
which  gives  the  most  pronounced  reaction  is  o.or  grm.  per  cent., 
while  that  which  gave  just  an  appreciable  reaction  was  0.00 1  grm.  per 
cent.  With  potassium  nitrate  no  attraction  could  be  detected  at  0.05 
grm.  per  cent.,  whereas  there  was  a  slight  one  at  o.  i  per  cent. 
Roughly  estimated,  therefore,  malic  acid  attracts  fifty  times  more 
strongly  than  potassium  nitrate.     Strong  solutions  repel. 

Attempts  have  been  made  to  elucidate  the  phenomena  of  chemotaxis 


138 


ARCHEGONIATES. 


by  means  of  the  theory  of  electrolytic  dissociation  of  solutions,  and 
with  some  success.  As  regards  the  spermatozoids  of  ferns,  Buller  has 
shown  that  in  the  case  of  some  compounds,  as  certain  salts  of  potas- 
sium and  malic  acid,  the  attraction  is  probably  due  to  certain  ions.  It 
is  not  to  be  assumed,  however,  that  a  chemotactic  stimulus  may  be 
given  only  by  ions,  for  certain  substances  which  are  not  dissociated 
have  been  found  to  exert  a  chemotactic  stimulus.  In  this  connection 
it  is  interesting  to  note  that  Pfeffer  found  that  the  spermatozoids  of 
mosses  are  attracted  by  cane  sugar,  which  does  not  attract  the  sperma- 
tozoids of  ferns. 


Fig.  53. —  Archcgonium  of  Onoclea  sensibiiis. — (After  Shaw.) 

A,  vertical  section  through  an  open  archegonium,  probably  within  ten  minutes  after  entrance  of  first 

spermatozoid;  an  unchanged  spermatozoid  is  inside  egg-nucleus. 

B,  vertical  section  of  venter  of  an  archegonium  containing  spermatozoids,  and  a  collapsed  egg  with  a 

spermatozoid  irithin  nucleus :  thirty  minutes. 

Although  malic  acid  exerts  a  strong  chemotactic  stimulus  upon  the 
spermatozoids  of  certain  ferns,  yet  from  the  foregoing  it  is  evident  that 
the  attraction  by  the  mucilaginous  substance  extruded  from  the  arche- 
gonium is  not,  of  course,  a  decisive  proof  that  malic  acid  compounds 
are  present  in  that  substance. 

Before  the  archegonium  opens  the  egg-cell  is  concave  on  the  upper 
side.  The  nucleus  is  also  flattened  or  concave ;  it  is  in  the  resting 
stage  and  may  contain  one  or  more  nucleoli.  Shaw  has  observed  that, 
in  living  sections,  the  egg  swells  as  soon  as  the  canal  is  cleared  of 
its  dissolving  contents,  and  fills  the  venter.  That  part  which  was 
previously  concave  now  forms  the  receptive  spot.  In  fixed  and  stained 
preparations  the  author  has  found  this  same  condition  of  the  egg-cell 


PTERIDOPMYTA. 


139 


when  the  neck-canal  contained  many  spermatozoids,  and  when  one  lay 
against  the  receptive  spot,  but  had  not  peneti'ated. 

On  entering  the  extruded  mucilaginous  substance  the  spermatozoids 
leave  their  vesicles  behind,  and  their  motion  is  retarded.  The  cork- 
screw spiral  is  drawn  out  and  the  number  of  turns  apparently  increased. 
The  forward  motion  of  the  spermatozoid  is  accompanied  by  a  rotation 
which  corresponds  to  the  pitch  of  the  screw. 

The  behavior  of  the  spermatozoid  after  entering  the  egg  can  be  fol- 
lowed only  in  properly  fixed  and  carefully  stained  sections.  Shaw 
found  that  in  all  prothallia  killed  within  an  hour  after  the  entrance  of 
the  spermatozoid  into  the  archegonium  the  egg-cells  were  in  a  collapsed 
condition,  being  concave  on  the  outside,  and  the  nucleus  conforming 
to  the  shape  of  the  cell  (Fig.  55,  A).  The  concavity  of  the  egg-cell 
occupies  the  position  of  the  receptive  spot.  This  condition  was 
regarded  by  Shaw  as  normal,  and  not  the  result  of  killing  reagents, 
since  in  the  living  condition  spermatozoids  were  seen  moving  freely  in 
the  cavity  above  the  egg.     I  quote  as  follows : 

There  are  reasons  to  believe,  however,  that  the  collapse  is  not  an  artificial 
plasmolysis,  but  that  it  takes  place  as  soon  as  the  spermatozoid  enters  the  egg. 
The  mature  egg  has  been  described  (for  the  other  species,  O.  struthiopteris 
(Campbell,  '95))  as  having  a  large  hyaline  receptive  spot.  The  concavity  of 
the  collapsed  egg  occupies  the  position  of  that  spot.  That  it  was  formed  before 
the  plants  were  killed  seems  evident  from  the  movement  of  a  number  of  sper- 
matozoids in  the  venter.  This  can  be  seen  in  the  living  plants.  That  the 
number  of  these  spermatozoids  is  large  is  shown  by  the  specimens  stained  and 
sectioned.  They  could  hardly  have  been  carried  into  the  venter  by  the  fixing 
agent,  for  those  in  the  canal  were  fixed  first,  in  the  extended  condition,  and 
those  in  the  venter  afterward  in  the  contracted  form.  From  the  evidence  at 
hand  it  appears  that  as  soon  as  the  egg  is  entered  by  a  spermatozoid  it  loses  its 
turgidity,  and  the  spermatozoids  which  come  into  the  venter  afterward  meet 
with  little  or  no  resistance  from  the  egg.  It  may  be  that  the  turgid  condition 
of  the  egg,  in  the  first  place,  offers  mechanical  facility  for  the  screw-like  sper- 
matozoid coming  through  the  narrow  base  of  the  neck  to  force  itself  into  the 
cytoplasm  of  the  receptive  spot,  and  that  the  plasmolytic  condition  of  the  egg 
afterward  deprives  the  following  spermatozoids  of  this  advantage,  and  protects 
the  egg  from  injury  or  from  multiple  fertilization  by  them. 

In  sections  made  from  material  killed  in  both  chrom-acetic  and 
chrom-osmic-acetic  acid  the  author  has  also  observed  in  many  cases 
the  collapsed  condition  of  the  egg-cell  as  described  by  Shaw.  Several 
preparations  were,  however,  especially  interesting  as  they  tend  to  throw 
some  doubt  upon  the  collapsed  condition  being  a  normal  occurrence. 
In  one  of  these  two  or  more  spermatozoids  had  entered  the  egg,  one  of 
which,  or  rather  its  nucleus,  had   partly  penetrated  the  egg-nucleus; 


140 


ARCHEGONIATES. 


the  others  lay  in  the  cytoplasm  of  the  receptive  spot  (Fig.  56,  C). 
(In  this  figure  one  of  the  spermatozoids  was  cut  in  sectioning,  so  that 
only  two  separate  pieces  of  it  are  shown,  the  other  parts  being  in  the 
next  section.)  The  nucleus  was  concave  above,  but  the  egg-cell  had 
not  collapsed.  It  remained  apparently  turgid,  having  been  only 
slightly  shrunken  uniformly  on  all  sides  by  the  reagents.  The  mem- 
brane of  the  egg  seemed  to  be  firm,  but  whether  it  was  anything  more 
than  a  plasma  membrane  I  was  unable  to  determine.  The  prothal- 
lium  from  which  this  preparation  was  made  was  killed  in  chrom-acetic 


Fig.  56. — Fusion  of  sperm  and  egg-nucleus.     C,  OnocUa 
struthiopteris  ;   D  and  E,  OnocUa  sensibilis. 

C,  vertical  section  of  egg  ;  two  spermatozoids  have  pene- 
trated egg,  one  of  which  is  just  entering  egg-nucleus  ; 
the  egg  is  globular,  but  its  nucleus  is  concave  above. 

D,  vertical  section  of  egg ;  outside  spermatozoids  are 
forced  against  venter  wall  by  expanding  egg;  sperm 
nucleus  within  egg-nucleus  has  begun  to  reticulate ; 
three  hours. 

E,  horizontal  sectional  section  of  an  egg;  fourteen  hours. 

(D  and  E,  after  Shaw.) 

acid,  and,  although  stained  on  the  slide  with  Bismarck  brown  in  addi- 
tion to  the  Flemming  triple  stain,  there  was  nothing  to  indicate  with 
any  certainty  a  cellulose  character  of  the  membrane.  Lying  in  the 
cytoplasm  near  the  nucleus  of  each  spermatozoid  was  a  delicate  thread 
which  seemed  to  be  the  blepharoplast.  The  cytoplasmic  reticulum 
was  somewhat  shrunken  from  the  membrane  of  the  egg  on»one  side. 
In  another  preparation  mentioned  in  a  preceding  paragiaph  the  open- 
ing of  the  neck  of  the  archegonium  was  apparently  closed  by  a  plug  of 
spermatozoids  after  one  had  entei-ed.     This  spermatozoid  lay  against 


PTERIDOPHYTA. 


141 


the  oval  surface  (^f  the  receptive  spot,  but  had  not  penetrated  the 
plasma  membrane.  It  had  apparently  untwisted  and  had  begun  to 
reticulate,  as  its  structure  was  somewhat  granular  or  lumpy  in  appear- 
ance. In  still  another  instance  the  spermatozoid  had  just  passed 
through  the  plasma  membrane  at  the  receptive  spot.  The  egg  was 
not  collapsed,  but  quite  turgid.  The  receptive  spot  was  distinguished 
from  the  rest  of  the  cytoplasm  only  by  the  presence  of  fewer  granules 
and,  perhaps,  a  little  looser  reticulum.  Other  eggs  were  observed  in 
a  turgid  condition  (the  archegonium  being  open),  into  which  no  sper- 
matozoid had  penetrated,  but  the  nucleus  was  concave  on  the  upper 
side.  It  may  be  mentioned  that  the  nucleus  is  not  always  concave, 
but  may  be  rounded  or  globular.  Apart  from  these  instances  the 
observations  of  the  author  agree  with  those  of  Shaw. 

In  about  one-half  hour,  or  less,  after  the  entrance  of  the  spermato- 
zoid into  the  archegonium,  the  canal  is  closed  by  the  expansion  of  the 
four  proximal  neck-cells  and  the  four  just  beyond  them.  The  egg 
recovers  its  turgidity  and  forces  the  free  spermatozoids  against  the 
outer  wall  of  the  venter  (Fig.  56,  D).  A  cellulose  membrane  does  not 
seem  to  be  formed  about  the  egg  immediately,  although,  as  stated  by 
Shaw,  a  very  delicate  cellulose  wall  may  have  been  dissolved  by  the 
chromic  acid  used  in  fixing.  Soon  after  penetrating  the  egg  the  nucleus 
of  the  spermatozoid  enters  the  egg-nucleus  before  undergoing  any 
change  in  form  or  visible  structure  (Fig.  55,  B).  The  fate  of  the 
cytoplasmic  part  was  not  very  satisfactorily  followed,  but  all  the  facts 
observed  indicate  that  the  cytoplasmic  band  and  blepharoplast  are  left 
in  the  cytoplasm  of  the  egg,  where,  as  in  Cycas  and  Zamia  of  the 
Gymnosperms,  they  are  absorbed.  In  Fig.  56,  D,  a  body  lying  near 
the  concave  side  of  the  nucleus  bears  some  resemblance  to  the  cyto- 
plasmic part  of  the  spermatozoid.  The  author  has  also  observed  in 
several  instances  undoubted  traces  of  the  blepharoplast  near  the  upper 
surface  of  the  nucleus,  and  there  is  no  question  but  that  the  fate  of 
the  blepharoplast  and  cytoplasm  is  as  just  stated. 

The  egg-nucleus  during  the  entire  process  of  fecundation  is  in  the 
resting  condition.  Several  conspicuous  nucleoli  are  usually  present. 
They  vary  in  size  and  have  a  vacuolate  structure.  In  the  delicate  linin 
network  are  distributed  the  small  chromatin  granules. 

In  a  short  time  the  sperm-nucleus  within  the  egg-nucleus  begins  to 
reticulate,  becoming  visibly  granular  and  of  a  looser  structure.  This 
is  apparent  three  hours  after  the  entrance  of  the  spermatozoid  into  the 
archegonium  (Fig.  56,  D),  but  it  may  sometimes  be  seen  earlier,  after 
thirty  minutes  or  one  hour.     The  time  after  which  a  change  is  notice- 


142  ARCHEGONIATES. 

able  in  the  sperm-nucleus  varies  greatly  in  different  individuals.  In 
some  cases  the  sperm-nucleus,  after  two  days,  showed  no  further 
advance  than  was  observed  in  others  after  only  thirty-six  hours.  As  the 
reticulation  of  the  sperm-nucleus  continues,  its  structure  becomes  looser 
and  more  open,  and  its  cork-screw  shape  disappears  (Fig.  56,  D,  E). 
As  far  as  is  known  at  present  the  reticulation  of  the  sperm-nucleus 
continues  until  its  network  is  no  longer  recognizable  from  that  of  the 
egg  when  fecundation  is  complete. 

During  the  process  of  fusion  it  will  be  seen  that  the  sperm-nucleus 
goes  through  the  same  series  of  changes  as  in  the  development  of  tlie 
spermatozoid,  but  in  the  reverse  order.  The  time  elapsing  between 
the  entrance  of  the  sperm-nucleus  into  the  egg  and  complete  fusion 
may  vary  considerably  in  individual  cases. 

\xi  Pilularia  globulifera^  according  to  Campbell  ('S8),  the  sperm- 
nucleus  assumes  a  loose  and  more  granular  structui'e,  and  rounds  up 
before  penetrating  or  uniting  with  the  nucleus  of  the  &%%.  Judging 
from  Campbell's  figures,  it  seems  that  in  Osmunda  (Campbell,  '92) 
the  sperm-nucleus,  as  in  Onoclea,  enters  the  nucleus  of  the  egg  before 
undergoing  any  visible  change  in  form  or  structure. 

In  this  respect  certain  ferns  are  without  parallel  in  the  plant  king- 
dom, except,  perhaps,  in  the  Gymnospei-ms,  and  it  would  be  inter- 
esting to  know  how  widely  distributed  the  phenomenon  is  in  the 
Pteridophyta^  and  whether  it  occurs  in  any  other  plants. 

GYMNOSPERMS. 
CYCAS,  ZAMIA,  AND  GINKGO. 

THE  MALE  GAMETOPHYTE. 

The  development  of  the  spermatozoid  in  Cycas  (Ikeno,  '96,  '98) , 
Ginkgo  (Hirase,  '96,  '98;  Webber,  '97;  Fujii,  1900),  and  Zamia 
(Webber,  '97,  1901),  bears  a  striking  resemblance  to  that  in  the  fern, 
especially  in  regard  to  the  origin  and  behavior  of  the  blepharoplast. 
There  seems  now  to  be  no  doubt  that  the  blepharoplast  in  these  three 
genera  is  homologous  to  the  blepharoplast  of  the  fern,  and,  in  fact,  the 
entire  development  of  both  sexual  cells  indicates  with  a  certainty  that 
these  gymnosperms  bear  a  close  phylogenetic  relationship  to  the 
pteridophytes. 

Since  the  development  of  the  spermatozoid  in  Cycas  and  Zamia 
differs  in  certain  important  details  according  to  the  two  investigators, 
Ikeno  and  Webber,  a  somewhat  detailed  account  of  the  process  will  be 
given  for  both  genera,  while  Ginkgo  will  be  referred  to  for  comparison. 


GYMNOSPERMS. 


H3 


The  mature  microspore  of  Cycas  revoluta,  according  to  Ikeno, 
consists  of  a  large  tube  cell  the  so-called  vegetative  cell,  which  gives 
rise  to  the  pollen  tube,  and  two  smaller  prothallial  cells  (Fig.  57,  A, 
;)„  A)-  The  nucleus  of  the  tube-cell  is  large,  and  contains  a  loose 
thread-work  and  a  nucleolus.  The  nuclei  of  the  prothallial  cells  are 
smaller,  and  flattened  to  conform  with  the  shape  of  those  cells.     The 


Fig.  57.— Microspore  and  development  of  male  gametophyte  in  Cycas  revoluta.—{Mtn  Ikeno.) 

A,  mature  microspore.    >,,  outer,/,,  inner  prothallial  cells;  ez,  tube  cell. 

B,  proximal  end  of  pollen  tube  capped  by  exineof  spore;  two  prothallial  cells, /i  and /,,  have  rounded 

off  and  increased  in  sire. 

C,  same  at  later  stage  of  development ;  the  inner  prothallial,  or  antheridial,  cell  has  divided  into  the 

generative  cell  iind  stalk  cell  {it);  >,,  first  prothallial  cell;  c,  c,  primordia  of  blepharoplast.s ; 
r,  nucleolus  of  generative  cell  nucleus. 

D,  later  than  C  ;  the  blepharoplast  primordia  {c)  have  moved  away  from  nucleus. 

E,  proximal  end  of  pollen  tube  shortly  before  division  of  generative  cell  (.kz)  which  has  increased 

greatly  in  size;  the  large  blepharoplasts  are  provided  with  beautiful  radiations  ;  the  tube  nucleus 
{ezk)  has  migrated  back  into  proximal  end  of  tube. 

walls  cutting  off  the  prothallial  cells,"  according  to  Ikeno,  are  straight, 
meeting  the  wall  of  the  pollen  spore,  while  in  Zamia  Webber  finds 
that  these  walls,  which  are  only  plasma  membranes,  are  arched  out 
into  the  tube  cell.  The  inner  cell  (/,)  gives  rise  to  the  antheridium, 
and  may  be  known  as  the  antheridial  cell. 

A  period  of  about  three  months  elapses  between  pollination,  which 
takes  place  early  in  July,  and  fecundation  in  October.     Immediately 


144  ARCHEGONIATES. 

after  pollination  each  spore  in  the  pollen  chamber  of  the  macrosporan- 
gium  germinates,  the  tube  cell  developing  gradually  into  a  branched 
tube  which  penetrates  the  tissue  of  the  nucellus.  The  tube-nucleus 
passes  into  the  tube,  maintaining  a  position  near  the  gi-owing  region 
or  end  as  long  as  the  tube  continues  its  growth  into  the  tissue  of  the 
nucellus,  while  the  two  prothallial  cells  retain  their  former  position. 
Contrary  to  the  genus  Pinus  and  other  higher  Conifers  the  distal  end 
of  the  tube  does  not  grow  directly  toward  the  archegonia,  but  later- 
ally and  downward,  serving  especially  as  an  organ  for  the  absorption 
of  food  (Fig.  65,  A).  The  proximal  end  of  the  tube,  carrying  before 
it  the  cap  of  exine,  or  the  remaining  outer  wall  of  the  spore,  finally 
grows  toward  the  archegonium.  The  pollen  tube  has  a  similar  beha- 
vior in  Zamia  (Webber,  '97)  and  Ginkgo  (Hirase,  '98). 

Soon  after  the  germination  of  the  spore  the  two  prothallial  cells 
increase  in  size,  especially  the  antheridial  cell,  which  becomes  spherical 
(Fig.  57,  B, /2)'  Its  nucleus  is  also  correspondingly  large,  and  the 
cytoplasm  presents  a  looser  structure.  In  the  meantime  the  anthe- 
ridial cell  divides,  the  daughter-nuclei  being  of  equal  size.  According 
to  Ikeno  ('98,  p.  172)  a  wall  is  not  formed  between  these  two  nuclei 
in  Cycas  revoluta.  One  of  them  now  increases  rapidly  in  size,  so  that 
it  occupies  nearly  the  entire  cavity  of  the  mother-cell,  while  the  other 
remains  small  and  is  crowded  out  as  a  naked  nucleus  (Fig.  1^7,  C,  D, 
j/) .  The  larger  cell  is  known  as  the  generative  cell  (Korperzelle  of 
the  German  literature)  and  gives  rise  to  two  spermatozoids ;  the  smaller 
cell  is  the  stalk  cell  (Fig.  57,  C,  D,  st). 

As  we  shall  see  later  Webber  finds  that  the  antheridial  cell  divides 
regularly  into  the  stalk  and  generative  cells,  but  the  plasma  membrane 
separating  the  two  cells  is  delicate,  and  the  stalk  cell  arches  over  the 
first  prothallial  cell  in  such  a  manner  as  to  give  the  appearance  of  the 
latter  being  nearly  enclosed  by  the  former  (Fig.  60,  F,  G).  It  is  pos- 
sible that  the  same  is  true  also  for  Cycas.  The  plasma  membrane, 
being  very  delicate,  may  have  been  overlooked  by  Ikeno,  for  the  posi- 
tion of  the  two  cells  is  such  as  to  make  it  appear  that  the  stalk  nucleus 
was  forced  out  of  the  mother-cell. 

Soon  after  this  stage  of  development  two  small  bodies  appear  in  the 
generative  cell  (body-cell),  lying  close  to  the  nucleus  and  on  opposite 
sides  (Fig.  57,  C,  c).  Ikeno  seems  to  be  of  the  opinion  that  the  two 
bodies,  which  he  calls  centrosomes,  are  derived  from  the  nucleus,  for 
the  reason  that  just  prior  to  their  appearance  outside  of  the  nucleus, 
objects  staining  similarly  appear  within  the  nucleus.  These  bodies, 
which  are  the  primordia  of  the  blepharoplasts,  move  away  from  the 


GYMNOSPERMS. 


H5 


nucleus  toward  the  periphery  of  the  cell  (Fig.  57,  D,  c).  With  fur- 
ther growth  the  generative  cell  with  its  nucleus  becomes  elliptical,  their 
major  axis  lying  parallel  with  the  longitudinal  axis  of  the  tube.  The 
two  primordia  of  the  blepharoplasts,  which  lay  previously  in  line 
parallel  with  the  transverse  axis  of  the  tube,  are  now  found  in  the  ends 
of  the  generative  cell.  About  each  there  soon  appear  beautiful  kino- 
plasmic  radiations,  giving  them  a  most  striking  resemblance  to  centre- 
spheres  with  large  centrosomes.  Later  in  the  period  of  development, 
or  about  the  middle  of  August  in  Japan,  the  young  blepharoplasts 
shift  their  position  again,  so  that  their  earlier  orientation  in  the  gene- 
rative cell  with  respect  to  the  axis  of  the  pollen  tube  is  resumed  (Fig. 
57,  E).  The  generative  cell  becomes  spherical,  and  the  kinoplasmic 
radiations  are  very  conspicuous. 

From  this  time  until  the  end  of  September,  or  about  one  and  one- 
half  months,  few  changes  manifest  themselves  in  the  generative  cell 
apart  from  an  increase  in  size.  This  period  in  the  development  is, 
therefore,  a  period  of  growth,  which  corresponds  to  a  similar  period 
in  the  development  of  the  archegonium,  and  at  the  end  of  which  all 
elements  have  reached  their  maximum  size  (Fig.  57,  E) .  The  diameter 
of  the  generative  cell,  which  contains  dense  cytoplasm,  is  about  0.14 
mm.,  and  that  of  the  nucleus  is  about  60  /x.  The  primordia  of  the 
blepharoplasts  have  also  increased  considerably  in  size ;  they  are  about 
15  /xin  diameter.  Apart  from  the  presence  of  one  or  more  vacuoles,  they 
are  rather  homogeneous  massive  bodies.  The  kinoplasmic  radiations 
are  still  beautifully  developed ;  they  seem  to  pass  over  gradually  and 
insensibly  into  the  alveolar  structure  of  the  cytoplasm. 

About  the  middle  of  September  the  tube  nucleus  begins  to  migrate 
toward  the  proximal  end  of  the  pollen  tube,  and,  by  the  end  of  the 
month,  this  nucleus,  the  generative,  stalk,  and  outer  prothallial  cells 
are  ail  in  the  proximal  end,  which  is  capped  by  the  exine  of  the  spore. 
It  may  be  mentioned  here  that  the  migration  of  the  tube  nucleus  into 
the  proximal  end  of  the  pollen  tube  seems  to  be  a  striking  confirmation 
of  the  doctrine  of  Haberlandt,  namely,  that  in  a  growing  cell  the 
nucleus  generally  takes  a  position  near  the  seat  of  constructive  activity. 
Since  the  proximal  end  of  the  tube  now  grows  toward  the  archegonium, 
and  as  growth  at  the  distal  end  ceases,  it  is  to  be  expected,  in  harmony 
with  the  theory  of  Haberlandt,  that  the  nucleus  which  presides  over 
this  growth  should  move  toward  the  region  of  that  activity.  Webber 
has  observed  the  same  behavior  of  the  tube  nucleus  in  Zamia. 

The  final  processes  which  now  take  place  in  the  male  gametophyte 
have  to  do  largely  with  the  development  of  the  two  spermatozoids 


146 


ARCHEGONIATES. 


from  the  generative  cell.     To  this  phase  of  development  Ikeno  has 
applied  the  term  spermatogenesis. 

As  soon  as  all  the  structures  mentioned  accumulate  in  the  proximal 
end  of  the  tube,  all  save  the  generative  cell  begin  to  disorganize  and 
finally  disappear.     What  this  disorganization  signifies,  Ikeno  remarks, 


FiG-  58. — Division  of  generative  cell  and  further  development  of  blepharoplasts  in  Cycas 
revoluta  — (After  Ikeno.) 

A,  generative  cell  with  nucleus  in  early  prophase  of  division  ;  chromatin  scattered  in  masses  of  granules. 

B,  same  with  nucleus  in  late  anaphase ;  each  blepharoplast  has  separated  into  a  mass  of  rods  from 

which  radiations  extend  ;  they  have  nothing  whatever  to  do  with  mitotic  spindle. 

C,  blepharoplast  of  B  more  highly  magnified. 

D,  cell-division  is  about  complete  ;  the  radiations  have  nearly  disappeared  from  the  mass  of  granules 

composing  blepharoplast. 

E,  two  spermatozoid  mother-cells,  the  one  on  the  right  in  outline;  the  ciliated  blepharoplast  has  made 

one  turn  about  the  cell ;  nuclear  beak  is  in  connection  with  ciliated  band. 

is  an  open  question,  but  it  seems  that  all  of  the  disorganized  elements 
contribute  to  the  nourishment  of  the  generative  cell. 

The  cytoplasm  of  the  generative  cell  now  assumes  a  coarse,  net-like 
structure,  and  the  nucleus  divides  (Fig.  58,  A,  B).  The  details  of 
this  division  will  not  be  dwelt  upon  further  than  to  state  that  the 
mitotic  spindle  arises  without  the  intervention  of  the  ceutrosphere-like 


GYMNOSPERMS. 


HI 


primordia  of  the  blepharoplasts  (Fig.  58,  B) .  This  is  true  for  Zamia^ 
according  to  Webber,  and  for  Ginkgo^  according  to  Hirase.  At  this 
stage  each  prinnordium  of  the  blepharoplast  is  transformed  into  a  group 
of  fine  rods  about  which  the  radiations,  although  not  so  pronounced, 
are  still  present  (Fig.  58,  C).  When,  however,  the  daughter  chromo- 
somes have  arrived  at  the  poles  of  the  spindle,  each  blepharoplast  has 
become  a  mass,  or  an  accumulation,  of  granules,  and  the  radiations 
can  scarcely  be  recognized. 

At  the  close  of  nuclear  division  each  daughter-nucleus  is  homo- 
geneous, presenting  a  small  number  of  nucleoli.  A  cell-plate  is  formed 
and  the  division  of  the  generative  cell  completed  (Fig.  58,  D).  The 
next  step  is  characterized  by  the  behavior  of  thq  mass  of  granules  of 
the  young  blepharoplast.  These  are  arranged  close  to  the  nucleus 
into  a  more  or  less  short  and  broad  band  whose  granular  nature  is  still 
evident.  Seen  in  profile  a  number  of  radiations  appear  extending  out 
from  the  band  toward  the  periphery  of  the  cell  (Fig.  59,  A).  These 
radiations  are  the  developing  cilia  of  the  spermatozoid.  Whether  the 
cilia  are  transformed  radiations,  or  arise  anew,  is  a  question.  Ikeno 
('98,  p.  180)  is  inclined  to  think  that  the  former  mode  of  origin  is  the 
more  probable. 

In  the  meanwhile  the  nucleus  develops  a  beak  which  becomes  con- 
nected with  the  ciliated  band  (Fig.  59,  A).  The  development  of  the 
nuclear  beak  and  the  arrangement  of  the  granules  into  a  band  take 
place  simultaneously,  so  that  it  is  not  known  which  phenomenon  is  of 
first  importance.  If  the  formation  of  the  beak  took  the  initiative,  then 
it  would  be  reasonable  to  suppose  that  the  direct  cooperation  of  the 
nucleus  in  the  development  of  the  band  is  indispensable.  In  Zamia^ 
according  to  Webber,  no  such  nuclear  beak  occurs  in  the  development 
of  the  spermatozoid.  Subsequent  to  this  stage  in  the  development  of 
the  band  its  granular  nature  is  no  longer  recognizable ;  it  appears  as  a 
thin  homogeneous  thread  (Fig.  59,  B).  The  further  behavior  of  the 
blepharoplast  seems  to  be  characteristic  of  spermatogenesis  in  Cycas^ 
Zamia^  and  Ginkgo.  The  ciliated  band  extends  itself  in  a  spiral 
which  ultimately  makes  five  turns  around  the  hemispherical  cell, 
always  remaining  near  its  surface  just  beneath  the  plasma  membrane. 
During  this  process  the  nucleus  increases  in  size  and  becomes  some- 
what pear-shaped.  Its  beak,  to  which  is  attached  apparently  one  end 
of  the  band,  increases  in  length  until  it  almost  reaches  the  surface  of 
the  cell  (Fig.  58,  E,  and  Fig.  59,  B).  The  free  end  of  the  band  con- 
tinues its  spiral  course  around  the  cell  a  short  distance  beneath  the 
plasma  membrane.     The  direction  of  the  spiral  is  parallel  with  the 


148 


ARCHEGONIATES. 


plane  of  division  of  the  generative  cell.  In  Fig.  58,  E,  which  repre- 
sents a  median  section  through  the  two  daughter-cells,  the  blepharo- 
plast  has  made  one  turn  around  the  cell.  The  cilia,  which  at  first  lay 
wholly  within  the  cytoplasm,  project  out  through  the  plasma  membrane 
as  the  band  approaches  the  surface  of  the  cell.  The  nuclear  beak, 
which  remains  in  close  contact  with  the  band  during  its  earlier  develop- 
ment, finally  becomes  separated  from  it  (Fig.  59,  C).  In  the  mature 
spermatozoid  the  blepharoplast,  as  already  stated,  makes  about  five 
turns  around  the  cell  counter  clock-wise.     As  is  evident  from  a  median 


Fig.  59. — Further  development  of  spermatozoid  in  Cycas  revoluia. — (After  Ikeno.) 

A,  part  of  spermatozoid  mother-cell  showing  nuclear  beak  in  contact  with  granular  blepharoplast  band. 

B,  spermatozoid  mother-cell ;  band-shaped  blepharoplast  longer,  one  end  being  applied  to  nuclear  beak. 

C,  later  stage  ;  blepharoplast  has  made  about  three  turns  about  the  cell ;  the  nuclear  beak  seems  to 

have  separated  from  ciliated  band. 

D,  nearly  ripe  spermatozoid  in  median  section.     Both  nucleus  and  cytoplasm  are  lobed  on  one  side  as 

if  constricted  by  blepharoplast,  which  describes  about  five  turns  around  the  hemispherical  cell. 

section,  the  mature  spermatozoid  consists  of  a  large  nucleus  completely 
surrounded  by  a  thin  layer  of  cytoplasm,  and  the  blepharoplast  lies  in 
a  depression  or  groove  (Fig.  59,  D).  As  a  result  both  cytoplasm  and 
nucleus  are  lobed,  thus  presenting  a  wavy  contour  in  section.  This 
phenomenon  seems  to  indicate  that  during  the  final  increase  in  size  of 
the  nucleus,  the  blepharoplast  acted  as  a  kind  of  constriction  upon  the 
anterior  end  of  the  cell.  The  same  is  true  in  both  Zamia  and  Ginkgo. 
In  the  mature  spermatozoid  the  cytoplasm  which  completely  surrounds 
the  nucleus  is  clearly  distinguishable.     As  will  be  seen  for  Zamia  and 


GYMNOSPERMS. 


149 


Ginkgo^  the  spermatozoid  of  Cycas^  as  has  been  pointed  out  for  the 
fern,  is  a  transformation  of  the  entire  mother-cell. 

The  development  of  the  spermatozoid  in  both  Ginkgo  and  Zamia 
closely  resembles  that  in  Cycas.  That  in  Zamia  differs,  however, 
according  to  Webber,  in  certain  important  details,  and  because  of  this 
fact  the  process  in  Zamia  will  be  given  also  in  some  detail.  Webber 
investigated  two  species  growing  in  Florida — Zamia  Jloridiana  and 
Z.  pu7nila. 

As  a  rule  the  mature  microspore  of  Zamia  consists  of  the  tube  cell 
and  two  prothallial  cells  (Fig.  60,  A).  Only  in  exceptional  cases  were 
evidences  of  a  third  cell  observed,  but  if  three  prothallial  cells  are 
formed  in  the  development  of  the  pollen  spore  as  is  claimed  for  Cycas^ 
the  first  is  generally  absorbed  before  the  spore  is  mature,  leaving  only 
a  trace  in  the  form  of  a  dark  line.  The  two  prothallial  cells  are  pro- 
vided with  only  a  plasma  membrane.  The  first  prothallial  cell  is  shaped 
like  a  plano-convex  lens  and  arches  out  into  the  second  prothallial 
cell.  The  second  prothallial  cell  is  attached  to  the  first  and  arches  out 
into  the  tube  cell  (Fig.  60,  A,  B).  This  is  especially  marked  during 
the  growth  of  the  pollen  tube.  The  nucleus  of  the  tube  cell  is  larger 
than  those  of  the  prothallial  cells,  and  of  the  latter  the  nucleus  of  the 
first  is  larger  than  that  of  the  second.  Very  soon  in  the  growth  of  the 
pollen  tube  the  second  or  antheridial  cell,  together  with  its  nucleus, 
greatly  exceeds  the  first. 

The  process  of  pollination,  which  occurs  in  Florida  in  January, 
brings  the  pollen  grains  into  the  pollen  chamber,  a  cavity  in  the  apex 
of  the  nucellus,  formed  by  the  disorganization  of  the  tissue  of  the 
latter.  Webber  ('01)  states  that  the  passage  of  the  pollen  grain 
through  the  micropyle  is  evidently  accomplished  by  suction. 

A  somewhat  mucilaginous  fluid  is  secreted  by  the  cells  which  sur- 
round the  micropyle,  and  a  drop  of  this  fluid  is  probably  protruded 
at  the  time  of  pollination.  The  fluid  disappears  later,  and  during  the 
formation  of  the  pollen  chamber  a  suction  is  formed  by  the  breaking 
down  of  the  cells  in  its  formation,  so  that  the  fluid,  together  with 
the  pollen  grains  that  may  be  held  in  it,  is  brought  down  into  the 
pollen  chamber. 

In  a  short  time  after  the  pollen  grains  have  been  brought  into  the 
pollen  chamber  they  germinate,  the  tube  bursting  out  of  the  exine  of 
the  grain  at  a  point  opposite  the  prothallial  cells  (Fig.  60,  B).  No 
matter  what  the  position  of  the  grain  may  be,  the  tube  always  pene- 
trates the  tissue  of  the  nucellus  adjacent  to  the  chamber.  The  tube  in 
Zamia  does  not  branch  before  entering  the  nucellar  tissue,  and  only 


ISO 


ARCHEGONIATfiS. 


occasionally  afterward  (Fig.  6^,  A).  During  the  early  development 
of  the  tube,  the  prothallial  cells  increase  in  size,  becoming  broader 
and  longer.  The  first  prothallial  cell  pushes  out  into  the  second, 
which  becomes  shaped  like  a  concavo-convex  lens,  and  is  crescent- 
shaped  in  cross-section  (Fig.  60,  B,  C).  As  stated  in  a  preceding 
paragraph,  the  behavior  of  the  tube  nucleus  is  similar  to  that  in  Cycas. 


Fig.  60. — Microspore  and  development  of  male  gametophyte  in  Zaniia. — (After  Webber.) 

A,  mature  pollen  grain  ;  at  point  of  attachment  of  the  two  prothallial  cells,  on  left,  a   dark  crescent- 

shaped  line  represents  a  layer  in  wall  of  spore,  which  may  be  the  remains  of  a  third  resorbed  pro- 
thallial cell. 

B,  germinating  pollen  grain,  early  stage.     The  two  prothallial  cells  have  not  yet  begun  to  increase 

in  size. 

C,  later  stage  of  germinating  pollen  graia  ;  the  tube  nucleus  has  increased  in  size  and  passed  out  into 

tube  ;  prothallial  cells  unchanged. 

D,  proximal  end  of  pollen  tube  ;  the  two  prothallial  cells  have  increased  in  size,  the  first  having  crowded 

out  into  the  second  in  a  marked  degree. 

E,  proximal  end  of  pollen  tube;  nucleus  of  second  prothallial  cell,  antheridial  cell,  in  telophase  of  divi- 

sion, lower  end  of  mitotic  figure  being  crowded  to  one  side  by  the  encroaching  first  prothallial  cell. 

F,  prothalliumin  proximal  end  of  tube,  after  division  of  antheridial  cell  into  stalk  and  generative  cell. 

G,  prothallium  in  later  stage  of  development  after  appearance  of  blepharoplasts  ;  the  double  plasma 

membrane,  separating  first  prothallial  cell  and  stalk  cell,  shows  that  there  are  two  distinct  and  inde- 
pendent cells  of  separate  origin. 

A  little  later  the  second  cell  has  arched  out  very  greatly,  and  the 
increase  in  size  of  the  first  prothallial  cell  has  brought  the  second,  or 
anthei'idial  cell,  out  beyond  the  limits  of  the  pollen  grain  and  into 
the  tube  (Fig.  60,  D).  However,  the  prothallium  remains  in  con- 
nection with  the  wall  of  the  pollen  spore  until  the  spermatozoids  are 
mature. 

The  next  important  step  in  the  development  is  marked  by  the 
division  of  the  second  prothallial  cell  into  the  stalk  cell  and  generative 


GYMNOSPERMS,  15I 

cell  (body  cell)  (Fig.  60,  E).  In  this  figure  the  division  is  in  the 
telophase,  the  two  daughter-nuclei  being  still  connected  by  the  con- 
necting fibres.  Owing  to  the  crescent  shape  of  the  cell  the  spindle 
lies  at  an  angle  to  the  major  axis  of  the  prothalHum,  the  lower  nucleus 
being  crowded  to  one  side  by  the  position  of  the  first  prothallial  cell, 
while  the  upper  nucleus  occupies  a  central  position  in  the  upper  half 
of  the  cell,  which,  when  the  wall  is  formed,  will  become  the  genera- 
tive cell  (body  cell,  central  cell).  The  lower  nucleus  becomes  the 
nucleus  of  the  stalk  cell.  Fig.  60,  F,  represents  the  next  stage  in 
which  the  division  is  complete.  A  distinct  transverse  plasma  mem- 
brane is  formed  just  above  the  apex  of  the  first  prothallial  cell  which 
is  almost  entirely  surrounded  by  the  stalk  cell.  It  is  clear  that  should 
the  plasma  membrane  separating  the  generative  from  the  stalk  cell  be 
very  delicate  and  somewhat  obscured,  the  nucleus  of  the  stalk  cell 
would  appear  to  be  forced  out  to  one  side.  For  this  reason  it  seems 
possible  that  the  plasma  membrane  separating  stalk  and  generative 
cells  in  Cycas  was  overlooked  by  Ikeno.  In  Ginkgo  the  first  prothal- 
lial cell,  which  according  to  Webber  is  also  surrounded  by  the  stalk 
cell,  was  considered  by  Hirase  ('98)  to  be  strands  of  cytoplasm  in  the 
second  prothallial  cell.  Miyake  ('02),  who  has  also  examined  Ginkgo^ 
confirms  the  observations  of  Webber. 

At  the  stage  of  Fig.  60,  F,  according  to  Webber,  the  nucleus  of  the 
generative  cell  is  9.79  y-  in  diameter,  that  of  the  stalk  cell  7.12  ;/,  while 
the  first  prothallial  cell  is  8.9  [i  in  diameter.  The  entire  prothalHum 
is  29.37  i^  ^0"g  by  16.91  (i  wide. 

Neither  during  the  division  of  the  second  prothallial  cell  into  stalk 
and  generative  cell  nor  for  some  time  afterward  was  anything  observed 
in  the  cell  in  connection  with  the  spindle,  or  elsewhere,  that  suggested 
a  young  blepharoplast.  It  is  not  until  the  generative  cell  has  increased 
considerably  in  size  that  the  first  traces  of  the  blepharoplasts  were  recog- 
nized. At  first  each  blepharoplast  consists  of  a  small,  deeply  staining 
granule,  from  which  several  filaments  of  kinoplasm  radiate,  following 
the  meshes  of  the  cytoplasmic  reticulum  (Fig.  60,  G).  "The  central 
granule  (Webber,  '01,  p.  31)  does  not  seem  to  be  different  in  sub- 
stance from  the  radiations — stains  the  same  and  shows  no  differentiation 
of  structure.  In  this  stage  it  is  only  a  half  micron  in  diameter  or  less, 
and  seems  to  be  scarcely  more  than  the  point  of  the  crossing  of  the 
filaments  of  kinoplasm.  These  granules  are  located  in  the  cytoplasm 
about  halfway  between  the  nucleus  and  the  cell- wall.  Two  are 
formed  in  each  central  cell  at  the  same  time  and  apparently  inde- 
pendently.    They  are   commonly  located  on   the  opposite   sides   of 


152 


ARCHEGONIATES. 


the  nucleus,  but,  in  a  number  of  cases  in  this  stage  and  in  a  still  later 
stage,  they  have  been  found  nearer  together,  frequently  less  than  45° 
apart." 

The  first  indication  of  a  differentiation  in  the  blepharoplast  as  it 
increases  in  size  is  seen  in  the  formation  of  an  outer  wall  or  membrane. 
The  generative  cell,  v^^hich  has  remained  nearly  spherical,  increases  in 


Fig.  61. — Prothallium  and  a  dividing  generative  cell  of  Zantia. — (After  Webber.) 

A,  prothallium  in  which  generaiive  Cell  has  become  large  and  elongated  ;  the  blepharoplasts  have  taken 

positions  on  opposite  sides  of  nucleus,  corresponding  to  longitudinal  axis  of  pollen  tube;   starch 
grains  have  begun  to  appear  in  stalk  cell. 

B,  division  of  generative  cell,  nucleus  in  anaphase,  showing  hyaline  cytoplasmic  areas  around  poles  ; 

the  blepharoplasts,  whose  outer  membranes  have  separated  into  pieces  or  segments,  are  not  con- 
nected with  spindle. 

size  and  becomes  elliptical  or  oblong,  its  major  axis  nearly  coinciding 
with  the  longitudinal  axis  of  the  pollen  tube  (Fig.  61,  A).  The 
blepharoplasts  by  this  time  have  taken  a  position  on  opposite  sides  of 
the  nucleus  on  the  line  of  the  major  axis  of  the  cell.  The  kinoplasmic 
radiations  are  slightly  more  prominent  than  the  lamellse  or  fibrillae  of 
the  cytoplasmic  reticulum  into  which  they  run  and  disappear  (Fig.  6 1 ,  A) . 


GYMNOSPKRMS. 


153 


About  the  first  of  April  the  blepharoplasts  have  reached  nearly  one- 
half  the  size  they  finally  attain.  They  are  more  or  less  vacuolate,  and 
the  kinoplasmic  radiations,  which  have  become  more  abundant,  extend 
in  many  instances  quite  to  the  plasma  membrane  of  the  cell. 

After  further  growth  the  generative  cell  divides  into  the  two  cells 
which  develop  into  the  two  spermatozoids  (Fig.  61,  B,  and  Fig.  62). 
The  blepharoplasts  take  no  part  in  the  division  of  the  nucleus.  Al- 
though their  kinoplasmic  radiations  become  fewer,  they  do  not  enter 
into  the  formation  of  the 
spindle,  as  the  latter  devel- 
ops apparently  entirely 
within  the  nucleus,  and  is 
almost  mature  before  the 
nuclear  membrane  has  dis- 
appeared. In  the  spindle 
stage  of  this  division  the 
blepharoplast  is  seen  to 
have  undergone  a  noticeable 
change.  It  has  increased  in 
size  and  its  outer  membrane 
has  separated  from  the  con- 
tents, which  are  somewhat 
shrunken.  The  outer  mem- 
brane has  separated  into 
fragments  or  plates,  and 
appears  now  as  a  broken 
line  (Fig.  61,  B).  The 
kinoplasmic  radiations  have 
almost  disappeared.  The 
reticulum  of  the  cytoplasm 
about  the  blepharoplast  is 
so  arranged  as  to  suggest 
radiations.  It  will  be 
remembered  that   precisely  the  same  phenomenon  occurs  in  Cycas. 

During  the  anaphase  of  division  the  finer  structure  of  the  outer 
membrane,  which  still  consists  of  a  number  of  segments,  is  seen  to  be 
made  up  of  numerous  small  granules  placed  side  by  side  to  form  the 
membrane.  The  central  contents,  which  stained  very  densely  at  an 
earlier  stage,  have  disappeared,  giving  place  to  a  delicate  hyahne 
reticulum  (Fig.  61,  B).  Webber  suggests  that  the  densely  stammg 
material  which  resembled  nucleoli  in  its  staining  qualities  was  utilized 


Fig.  62.— Prothallium  of  Zamia  in  which  the  generative 
cell  has  divided. 

The  blepharoplasts  have  separated  into  granules  which  are 
beginning  to  organize  the  ciliferous  band.  The  first 
prothallial  cell  and  stalk  cell  have  become  gorged  with 
starch.  (The  magnification  of  this  figure  is  only  one- 
half  that  of  A,  Fig.  61.)— (After  Webber.) 


154 


ARCHEGONIATKS. 


as  food  material  in  the  growth  of  the  blepharoplasts  and  other  parts 
of  the  cell.  During  the  telophase  the  blepharoplast  is  represented  by 
a  more  or  less  irregular  or  spherical  mass  of  granules,  which  have  evi- 
dently been  derived  by  the  breaking  up  of  the  membrane.  "It  would 
seem  that  the  outer  membrane  of  the  blepharoplast  breaks  up  into 
numerous  segments  or  granules,  which  assume  a  roundish  or  elliptical 
form,  and  through  the  action  of  the  cytoplasm  become  crowded  to- 
gether in  a  mass  occupying  the  position  of  the  original  blepharoplast." 
About  the  time  of  the  reconstruction  of  the  daughter-nuclei  and  the 
formation  of  the  plasma  membranes  separating  the  cells,  the  develop- 


FiG.  63. — Further  development  of  blepharoplast. — (After  Webber). 

A,  two  attached  spermatozoid  mother-cells  (spermatids)  resulting  from  division  of  generative  cell; 

the  band  of  blepharoplast  is  being  formed  by  fusion  of  granules. 

B,  fusion  of  granules  to  form  the  band. 

C,  formation  of  ciliferous  band  by  fusion  of  granules,  more  highly  magnified. 

ment  of  the  band,  which  is  to  bear  the  cilia,  begins.  It  appears  first 
as  a  short,  delicate,  and  deeply  staining  line  extending  from  the  mass 
of  granules  toward  the  nucleus  (Fig.  63,  A).  A  little  later  a  similar 
line  or  band  can  be  seen  on  the  opposite  side  of  the  mass  of  granules. 
From  Fig.  63,  B,  it  is  apparent  that  the  band  is  developed  more  or 
less  directly  from  the  granules.  The  band,  which  at  first  is  very  nar- 
row, increases  appreciably  in  width  (Fig.  63,  B,  C).  The  further 
development  of  the  band  with  its  cilia  and  the  transformation  of  the 
daughter-cell  into  a  spermatozoid  closely  resembles  that  of  Cycas, 
already  discussed  at  some  length  in  the  preceding  pages,  with  the  very 
noteworthy  exception  that  in  Zamia  there  is  no  nuclear  beak  formed, 


GYMNOSPERitS.  I55 

which  is  in  contact  with  one  end  of  the  blepharoplast  in  the  earlier 
part  of  its  development  (Fig.  63,  A). 

The  mature  spermatozoid  is  also  quite  similar  in  structure  to  that  of 
Cycas,  consisting  of  a  large  nucleus  completely  surrounded  by  a  layer 
of  cytoplasm  in  which  the  ciliferous  band,  or  blepharoplast,  is  located 
just  beneath  the  plasma  membrane.  The  blepharoplast  is  in  the  form 
of  a  helicoid  spiral,  making  about  five  or  six  turns  counter  clock-wise 
and  embracing  about  one-half  of  the  body  of  the  cell  (Fig.  65,  B). 
The  spermatozoid,  as  in  the  ferns,  is  a  transformation  of  the  entire 
cell  and,  therefore,  a  true  spermatozoid. 

The  development  of  the  spermatozoid  in  Ginkgo  according  to  Hirase 
('98)  is  quite  similar  to  that  in  Cycas  as  described  by  Ikeno.  In  the 
generative  cell  of  Ginkgo  Webber  ('97)  and  Hirase  ('98)  find  that, 
when  the  nucleus  becomes  strongly  flattened  or  lenticular,  a  large 
nucleolus-like  body  appears  on  either  side  of  the  nucleus  between  the 
nuclear  membrane  and  the  young  blepharoplasts.  Other  similar  but 
smaller  bodies  are  sometimes  present  in  the  cell.  Accompanying  these 
two  bodies  Hirase  finds  coarsely  granular  cytoplasm.  The  bodies  in 
question  react  toward  stains  much  as  do  nucleoli,  and,  since  they  dis- 
appear at  a  later  stage,  it  is  probable  that  they  represent  merely  extra- 
nuclear  nucleolar  substance. 

Miyake  ('02)  finds  that  after  the  division  of  the  generative  cell  in 
Ginkgo  a  cell-wall  is  formed  between  the  two  daughter-cells,  and  that 
a  distinct  and  firm  wall  was  always  found  around  the  two  spermato- 
zoids.  The  fact  that  a  wall  is  or  is  not  formed  about  the  daughter- 
cells,  /.  e.,  the  mother-cells  of  the  spermatozoids,  does  not  affect  the 
morphological  rank  of  the  spermatozoid. 

The  mature  spermatozoid  of  Zamia  is  probably  the  largest  male 
gamete  known  in  the  plant  kingdom,  being  plainly  visible  to  the 
unaided  eye.  When  swimming  freely  and  without  pressure  it  is 
slightly  ovate,  nearly  round  or  compressed  sphexical  (Fig.  65,  B). 
They  vary  greatly  in  size,  however,  ranging  in  length  from  222  to 
332  ;u,  and  in  width  from  222  to  306  p.. 

Ikeno  describes  the  spermatozoid  of  Cycas  as  being  provided  with 
a  tail  which  is  merely  the  elongation  of  the  posterior  part  of  the  cyto- 
plasmic mantle.  Measured  in  sections  the  length  was  found  to  be  160// 
and  the  width  70  //.  The  length  of  the  tail  was  80  yu  or  equal  to  that 
of  the  body.  Fujii  has  shown  that  the  tail  attributed  to  the  spermato- 
zoid of  Ginkgo  was  an  artifact,  and  this  statement  has  been  confirmed 
by  Miyake,  Since  no  tail  exists  in  Zamia^  it  is  probable  that  that 
described  for  Cycas  may  also  have  been  the  result  of  abnormal 
conditions. 


156  ARCHEGONIATES. 

THE  ARCHEGONIUM. 

The  development  of  the  archegouium  in  the  Cycadacece  and  in 
Ginkgo^  which  is  similar  to  that  of  Plnus,,  is  too  well  known  to  require 
a  detailed  description  in  this  place.  The  manner,  however,  in  which 
the  large  central  cell  is  nourished  during  its  growth  by  the  immediately 
surrounding  cells  of  the  prothallium  is,  if  Ikeno's  observations  be  cor- 
rect, a  phenomenon  of  a  rather  rare  occurrence  in  the  Gymnosperms, 
and  merits  some  special  mention.  These  surrounding  cells,  which  are 
separated  from  the  central  cell  by  thick  cellulose  walls,  are  of  a  uniform 
size,  each  possessing  dense  cytoplasm  and  a  large  nucleus.  Before 
the  archegonium  is  full  grown  the  nuclei  of  these  cells  show  a  fine  and 
distinct  threadwork ;  but,  as  this  organ  approaches  maturity,  the 
nuclei,  with  the  exception  of  the  nucleoli,  are  transformed  into  homo- 


FlG.  64. — Three  cells  from  layer  of  prothallial  cells  immediately  surrounding  upper  part  of  central  cell 
of  archegonium  of  Cycas,  showing  protoplasmic  connections  between  these  cells  ;  in  B  the  bealc  of 
nucleus  extends  into  plasmic  bridge. — (After  Ikeno.) 

geneous  and  diffusely  staining  bodies.  This  phenomenon  is  not  confined 
solely  to  the  cells  forming  the  wall  of  the  archegonium,  but  it  may 
extend  to  adjacent  cells  of  the  prothallium.  This  nuclear  change  takes 
place  only  in  cells  near  the  upper  part  of  the  central  cell. 

Goroschankin  has  shown  that  in  the  Cycadacece  fine  cytoplasmic 
connections  exist  between  the  central  cell  of  the  archegonium  and  the 
surrounding  cells.  Fi'om  Ikeno's  figures  it  seems  that  the  cytoplasmic 
strands  in  Cycas  are  relatively  large,  and  that  large  granular  plasmic 
masses  pass  over  bodily  into  the  central  cell  (Fig.  64,  A,  B).  Fre- 
quently the  nucleus  itself  will  send  out  a  beak  or  protuberance  toward 
the  nearest  plasmic  connection.  Arnoldi  (1900)  finds  that  in  several 
species  of  Pinus  and  in  Abies  the  nuclei  from  the  surrounding  cells 
pass  into  the  egg-cell.  The  prevalence  of  condensed  nuclei  in  cells 
surrounding  the  upper  part  of  the  central  cell  is  explained  by  Ikeno  as 


GYMNOSPERMS. 


157 


being  due  to  a  greater  need  of  food  material  by  this  part  of  the  central 
cell ;  for  it  is  here  that  the  greatest  activity  takes  place  during  the 
maturing  of  the  egg-cell,  which  culminates  in  the  formation  of  the 
ventral  canal-cell.  Webber  does  not  find  any  protoplasmic  connections 
between  the  egg-cell  and  those  surrounding  it  in  Zamia^  and  so  far 
as  the  author  is  aware  no  such  protoplasmic  connections  exist  in  the 
higher  Gymnosperms.  In  Cycas  the  phenomenon  described  by  Ikeno 
is,  if  true,  probably  an  adaptation  to  the  rapid  transfer  of  nutritive 
material  from  the  surrounding  cells  to  the  egg-cell. 

Strasburger  ('01,  pp.  550-553),  in  a  late  publication  on  the  proto- 
plasmic connections  between  cells  in  plants,  calls  into  question  the 
statement  that  nuclei  or  nuclear  fragments  pass  bodily  through  the  pits 
of  the  surrounding  cells  into  the  egg-cell  of  Gymnosperms  as  a  normal 
phenomenon,  and  asserts  that  it  is  the  result  of  injury  due  to  pressure 
or  fixing  reagents. 

There  seems  to  be  no  doubt  that  in  all  Gymnosperms  in  which  the 
egg-cells  reach  such  an  enormous  size  the  cells  immediately  surround- 
ing the  &g^  contribute  directly  to  the  nutrition  of  the  latter,  but  it  is 
not  clear  why  any  of  the  material  should  pass  over  bodily  into  the 
egg-cell. 

The  final  step  in  the  development  of  the  archegonium  is  the  forma- 
tion of  the  ventral  canal-cell,  which  takes  place  immediately  preceding 
fecundation,  and  consequently  this  cell  persists  only  a  short  time  (Fig. 
67,  A).  It  was  probably  due  to  this  fact  that  the  presence  of  a  ventral 
canal-cell  was  not  observed  by  Warming  and  Treub.  Only  a  plasma 
membrane  and  not  a  cell-wall  is  formed  separating  the  ventral  canal- 
cell  from  the  egg.  It  is  not  at  all  improbable  that  in  some  cases  a 
plasma  membrane  may  not  be  formed,  and  such  is  reported  for  Cefh- 
alotaxis  fortuni  by  Arnoldi  (1900).  The  formation  of  a  plasma 
membrane  is,  however,  of  secondary  importance  in  the  formation  of 
the  ventral  canal-cell,  for  if  the  nucleus  of  the  central  cell  of  the 
archegonium  divides  karyokinetically,  and  one  of  the  daughter-nuclei 
becomes  the  functional  egg-nucleus,  the  division  is  certainly  to  be 
regarded  as  the  formation  of  a  ventral  canal-cell  whether  a  plasma 
membrane  is  formed  or  not. 

Botanists  have  sometimes  been  inclined  to  refer  to  the  formation  of 
the  ventral  canal-cell  as  a  maturation  process  similar  to  that  in  the 
animal  egg.  Ikeno  speaks  of  this  step  in  the  development  as  the  period 
of  maturation  (Reifungsperiode),  which  recalls  the  formation  of  the 
polar  bodies  in  the  animal  egg,  but  I  do  not  infer  that  he  considers  the 
two  processes  homologous.     He  states,  however,  that  it  appears  prob- 


.58 


ARCHEGONIATES. 


able,  judging  from  the  karyokinetic  figures  observed,  that  the  nuclear 
division  leading  to  the  formation  of  the  ventral  canal-cell  is  of  the 
heterotypic  type,  and  takes  place  essentially  as  in  the  first  division  of 
the  pollen  mother-cells  of  the  LiliacecE.  This  is  certainly  an  error, 
for  in  both  Gymnosperms  and  Angiosperms  the  heterotypic  nuclear 
division  occurs  in  the  micro-  and  macrospore  mother-cells  and  nowhere 
else  in  ontogeny.  Since  the  spore  mother-cells  of  the  Gymnosperms 
are  homologous  with  those  of  the  higher  plants,  we  naturally  expect 
to  find  the  heterotypic  division  in  Cycas  in  the  first  karyokinesis  of 
the  macrospore  mother-cell.  This  is  made  all  the  more  certain  by  the 
researches  of  Juel  (1900),  who  finds  in  Larix  that  the  first  nuclear 
division  in  the  macrospore  mother-cell  is  heterotypic.     In  Larix  and 


Fig.  65. — Upper  end  of  nucellus  ;  spetmatozoids  in  pollen  tube  of  Zamia. — (After  Webber). 

A,  diagrammatic  outline  of  upper  end  of  nucellus,  showing  proximal  ends  of  pollen  tubes  growing  down 

into  the  cavity  just  above  archegonia ;  a,  archegonia ;  /,  prothallium ;  pc,  pollen  chamber ;  pt,  pol- 
len tubes;  pg,  pollen  grain. 

B,  two  mature  spermatozoids  in  proximal  end  of  pollen  tube. 

in  other  Gymnosperms  the  earlier  development  of  the  macrospore  is 
precisely  the  same  as  in  such  Angiosperms  as  Helleborus^  in  which 
the  first  nuclear  division  is  heterotypic  and  homologous  with  the  first 
division  in  the  pollen  mother-cell. 

The  formation  of  the  ventral  canal-cell  may  represent  some  sort  of 
a  maturation  process,  and  the  conclusion  that  this  cell  is  an  aborted 
&%%  is  tempting,  but  at  our  present  state  of  knowledge  such  an  infer- 
ence is  scarcely  justifiable. 

FECUNDATION. 

Soon  after  its  formation  the  ventral  canal-cell  disorganizes.  The 
nucleus  of  the  &^^  passes  back  gradually  toward  the  middle  of  the  cell, 
at  the  same  time  increasing  in  size.  Finally,  when  the  center  of  the 
cell  is  i-eached,  the  nucleus  is  usually  large,  being  generally  longer 
than  broad,  and  shows  the  structure  of  the  resting  condition. 


GYMNOSPKRMS. 


'59 


During  the  final  stages  in  the  development  of  the  spermatozoid  the 
proximal  end  of  the  pollen  tube,  which  is  still  capped  by  the  exine  of 
the  spore,  grows  downward  into  the  prothallial  cavity  as  in  Zamia 
(Fig.  65,  A).  This  cavity  in  Cycas,  according  to  Ikeno,  is  filled 
with  a  watery  fluid  derived  largely  from  the  archegonia,  and  in  which 
the  spermatozoids  swim  on  escaping  from  the  pollen  tube.  Webber 
is  of  the  opinion  that  in  Zamia  this  fluid  is  derived  largely  from  the 
pollen  tube. 

The  spermatozoids  in  Cycas,  on  escaping  from  the  pollen  tube,  swim 
about  rapidly,  and  in  a  short 
time  penetrate  the  ^%^. 
That  part  of  the  egg  at  which 
a  spermatozoid  enters  is  de- 
pressed, giving  the  impres- 
sion that  it  came  against  the 
egg  with  some  force.  The 
nucleus  of  the  spermatozoid 
now  escapes  from  its  cyto- 
plasmic mantle  and  migrates 
toward  the  nucleus  of  the 
egg.  The  cytoplasm  and 
blepharoplast  are  left  in  the 
upper  part  of  the  G%'g  as  in 
Zamia  (Fig.  ^G^  A,  B), 
where  they  undergo  disor- 
ganization. It  frequently 
happens  that  several  sperm- 
atozoids reach  the  ^%%^  but, 
as  a  rule,  only  one  penetrates 
into  its  interior,  the  others 
remaining  at  the  surface. 
Whether  more  than  one  male  nucleus  ever  fuses  with  the  egg-nucleus 
is  not  known. 

When  male  and  female  nuclei  come  in  contact  they  are  readily 
distinguished  from  each  other,  the  male  being  smaller,  with  a  more 
finely  granular  threadwork.  Both  are  in  the  resting  stage.  The  male 
nucleus  seems  to  pi-ess  against  the  female,  forming  a  depression  in  the 
latter.  In  a  short  time  the  male  nucleus  is  completely  imbedded  within 
the  egg-nucleus ;  the  membrane  of  the  male  nucleus  disappears,  and 
the  two  nuclei  fuse  so  completely  that  the  fusion  nucleus  can  scarcely 
be  distinguished  from  an  unfecundated  nucleus  of  the  egg. 


Fig.  66. — Fecundation  of  egg-cells  in  Zamia  — (After 
Webber.) 

A,  egg-cell  immediately  after  coming  together  of  male  and 

female  nuclei ;  the  ciliferous  'band  of  fecundating  sper- 
matozoid lies  in  upper  end  of  egg ;  a  second  spermato- 
zoid trying  to  gain  entrance  is  shown  at  apex  of  egg. 

B,  similar  to  A,  but  showing  longitudinal  section  of  ciliferous 

band  in  upper  end  of  egg. 


l6o  ARCHKGONIATES. 

The  processes  incident  to  and  accompanying  fecundation  in  Zamia 
differ  only  in  minor  details  from  those  of  Cycas.  Cei'tain  phases  of 
these  processes,  however,  as  observed  by  Webber  ('97,  I,  II,  III),  are 
of  special  interest  and  importance.  They  are  described  as  follows 
('97,  II,  p.  18): 

The  proximal  ends  of  the  pollen  tubes  .  .  .  which  grow  downward 
through  the  apical  tissue  of  the  nucellus  into  a  cavity  formed  in  the  prothallium 
above  the  archegonium,  have  increased  in  length  until  the  ends  almost  or  quite 
touch  the  neck  cells  of  the  archegonia,  which  protrude  into  the  same  cavity 
(Fig.  65,  A).  It  is  interesting  to  note  that  the  pollen  tubes  when  they  enter  the 
prothallium  cavity,  which  is  filled  with  air,  do  not  grow  at  random,  but  bend 
slightly  outward  and  grow  directly  toward  the  archegonia.  .  .  .  The  pro- 
truding tip  formed  by  the  old  pollen  grain  is  plainly  visible  with  a  hand  lens, 
and  is  evidently  the  point  which  first  comes  into  contact  with  the  neck  cells  of 
the  archegonia.  The  neck  cells  are  also  distended  and  turgid,  and  are  evi- 
dently easily  broken.  If  in  this  stage  the  end  of  a  pollen  tube  be  touched  very 
lightly  with  the  flat  side  of  a  scalpel  it  bursts,  and  the  antherozoids,  together  with 
a  drop  of  the  watery  contents  of  the  pollen  tube  are  quickly  forced  out,  and  the 
pollen  tube  immediately  shrivels  up  into  a  shapeless  mass.  .  .  .  The  pollen 
tube  evidently  grows  down  until  the  end  is  forced  against  the  neck  cells,  when 
the  tube  bursts,  discharging  the  mature  antherozoids  and  the  watery  contents 
of  the  tube  which  supplies  a  drop  of  fluid  in  which  the  antherozoids  can  swim. 
.     .     .     ('97,  III,  p.  226). 

As  explained  in  my  previous  papers,  several  antherozoids  commonly  enter 
each  archegonium,  two  being  usually  found  and  sometimes  three  or  four.  The 
entire  antherozoid  enters  unchanged,  swimming  in  between  the  ruptured  neck 
cells.  Only  one  of  the  antherozoids  is  concerned  in  fecundation,  and  the  others 
are  usually  found  between  the  protoplasm  and  the  wall  of  the  archegonium, 
presenting  their  original  form  and  appearance,  or  in  some  stage  of  disintegra- 
tion (Fig.  66,  A).  Occasionally  one  of  the  antherozoids  not  concerned  in  fecun- 
dation pushes  for  a  short  distance  into  the  contents  of  the  archegonium,  as  it  is 
always  found  in  such  cases  to  form  a  distinct  body  which  stains  very  differently 
.  .  .  ('01,  p.  65).  That  one  which  is  utilized  in  fecundation  swims  into  the 
protoplasm  of  the  archegonium  for  a  short  distance,  where  it  comes  to  rest  and 
undergoes  change.  The  nucleus  slips  out  of  its  cytoplasmic  sheath  and  passes 
on  alone  from  this  point  to  the  egg-nucleus,  with  which  it  unites.  The  spiral 
ciliferous  band  remains  at  the  apex  of  the  egg-cell  in  the  place  where  the  nucleus 
left.  In  very  numerous  instances,  just  after  fecundation,  it  has  been  discovered 
in  this  position,  and  there  can  be  no  doubt  that  this  process  is  the  one  normally 
occurring.  It  shows  very  plainly  and  presents  nearly  the  original  form  of  the 
spermatozoid  (Fig.  66,  B),  but  is  always  stretched  out  much  more  than  in  the 
normal  spermatozoid.     .     .     . 

The  method  of  escape  of  the  nucleus  from  the  body  of  the  spermatozoid  can 
only  be  conjectured.  It  would  seem,  however,  that  the  rapid  boring  of  the 
apical  or  spiral  end  into  the  egg-cell  may  cause  too  great  a  pressure  on  the 
large  body  of  the  spermatozoid,  resulting  in  its  bursting  and  freeing  the  nucleus , 


GYMNOSPERMS.  l6l 

while  the  cilia  motion  continues  probably  some  time  longer,  carrying  the  band 
farther  along  and  freeing  the  nucleus  from  any  hindrance  by  it.  The  apex  of 
the  spiral  end  of  the  spermatozoid  invariably  enters  the  egg-cell  first,  and  in  all 
of  the  cases  observed  where  the  nucleus  has  just  escaped  from  the  spermatozoid 
it  has  been  found  a  short  distance  behind  the  spiral  of  the  spermatozoid,  as  if 
it  had  been  forced  out  and  left  behind.  The  function  of  the  cytoplasm  of  the 
spermatozoid  is  still  in  considerable  doubt,  but  that  it  fuses  with  the  cytoplasm 
of  the  egg-cell  is  certain.  Shortly  after  the  nucleus  has  broken  out  of  the  sper- 
matozoid cell,  the  thin  layer  of  dense  cytoplasm  which  surrounded  it  can  be 
seen  in  a  broken,  fragmentary  form,  still  somewhat  connected  with  the  spiral 
band.  The  cytoplasm  of  the  spermatozoid  in  this  stage  is  very  different  from 
that  of  the  egg-cell,  being  more  densely  granular  and  staining  more  deeply,  so 
that  it  is  easily  distinguished.  Later,  only  a  coarse  granular  substance  is  found 
inside  the  spiral  coil  of  the  ciliferous  band,  and  it  would  seem  that  this  is  the 
cytoplasmic  matter  from  the  spermatozoid  which  has  mingled  with  that  of  the 
egg-cell.  It  should  be  mentioned  that  the  plasma  membrane  surrounding  the 
spermatozoid  has  entirely  disappeared,  no  trace  of  it  being  visible.  It  would 
seem  to  have  fused  with  some  substance  of  the  egg-cell  or  to  have  been 
absorbed  in  some  way. 

The  male  nucleus,  when  it  has  escaped  from  the  spermatozoid  and  is  observed 
lying  in  the  cytoplasm  at  the  apex  of  the  egg-cell,  is  a  loose,  open  structure, 
seeming  to  have  but  little  kinoplasmic  and  chromatin  matter.  The  passage  to 
the  nucleus  is  evidently  a  rapid  one,  as  few  stages  have  been  found  between 
the  above  and  the  completion  of  fecundation.  In  some  instances  the  path  over 
which  the  nucleus  travelled  in  reaching  the  egg-nucleus  is  discernible  by  the 
arrangement  of  the  granules  in  the  cytoplasm,  showing  the  direction  of  the 
passage. 

The  egg-nucleus,  previous  to  fecundation,  is  elliptical  and  is  located  slightly 
below  the  center  of  the  enormous  egg-cell  which  is  about  3  mm.  long  by  1.5  mm, 
wide  (Fig.  66,  A,  B),  The  egg-nucleus  is  of  enormous  size,  comparatively, 
being  plainly  visible  to  the  unaided  eye.  It  is  composed  of  an  open,  coarse 
reticulum.  So  far  as  the  writer  has  observed  there  is  no  depression  or  '•  emp- 
fangnisshohle  "  in  the  upper  part  of  the  nucleus  where  the  sperm-nucleus  enters, 
as  was  found  by  Ikeno  in  Cycas.  No  special  attention  has  been  given  to  this 
matter,  however,  and  further  observation  may  show  it  to  be  present.  The  male 
nucleus  in  entering  the  egg-nucleus  gradually  pushes  into  it  as  observed  by 
Ikeno  in  Cycas,  and  finally  becomes  entirely  surrounded  by  it.  Meanwhile  it 
has  changed  its  structure  and  become  densely  granular,  differing  markedly 
from  the  egg-nucleus  in  this  particular.  ,  ,  .  After  fecundation  is  apparently 
completed  the  male  nucleus  appears  as  a  small,  nearly  round  body  in  the  upper 
portion  of  the  egg-nucleus  into  which  it  has  penetrated  (Fig.  f^,  B). 

Further  changes  in  the  sexual  nuclei  were  not  followed  by  Webber, 
and  it  is  not  known  whether  a  fusion  nucleus  is  formed  in  Zamia  as 
described  by  Ikeno  for  Cycas. 

Since  the  publication  of  his  paper  on  Cycas,  Ikeno  ('01)  has  observed 
the  formation  of  the  ventral  canal-cell,  the  process  of  fecundation  and 


l62 


ARCHEGONIATES. 


the  first  division  of  the  fusion  nucleus  in  Ginkgo  biloba  (Fig.  67,  A, 
B,  C,  D).  These  processes  agree  closely  with  those  in  Cycas.  In 
Ginkgo^  however,  the  male  nucleus  at  the  time  of  fusion  is  relatively 
small,  being  less  than  one-tenth  the  size  of  the  female  nucleus.  As  in 
Cycas  and  Zamia^  the  male  nucleus  becomes  completely  imbedded  in 


Fig.  67. — Formation  of  ventral  canal-cell,  fusion  of  sexual  nuclei,  and  the  division  of  the  fusion  nucleus 

in  Ginkgo. — (After  Ikeno.) 

A,  apex  of  central  cell  of  archegonium  showing  telophase  of  nuclear  division  ;  a  cell-plate,  or  plasma 

membrane,  is  formed  in  the  connecting  fibers. 

B,  egg-nucleus  into  which  a  male  nucleus  {ni)  has  penetrated. 

C,  fusion  nucleus  in  prophase  of  division. 

D,  fecundated  egg-cell  showing  fusion  nucleus  in  spindle  stage  of  mitosis  ;  the  mitotic  figure  lies  within 

limits  of  the  nucleus  whose  membrane  seems  to  be  still  intact. 

the  female  before  the  dissolution  of  its  membrane.     Both  nuclei  are 
in  the  resting  condition  at  the  time  of  fusion. 

The  spindle  of  the  first  karyokinesis  following  fusion  is  formed 
within  the  nuclear  cavity  and  before  its  membrane  has  disappeared 
(Fig.  67,  B,  C,  D).  Nothing  is  said  by  Ikeno  about  being  able  to 
distinguish  male  and  female  chromatin  elements  in  this  division. 


GYMNOSPERMS.  163 

It  is  interesting  to  note  further  that  in  neither  Cycas,  Zamia^  nor 
Ginkgo  was  the  stalk  or  prothallial  cell  of  the  pollen  tube  found  in 
the  egg  by  any  of  the  observers  mentioned.  These  cells  are  probably 
disorganized  beyond  recognition  when  the  contents  of  the  tube  are 
discharged  into  the  egg. 

PINUS. 

THE  MALE  AND  FEMALE  GAMETOPHYTES. 

Apart  from  the  absence  of  motile  spermatozoids  and  the  behavior 
of  the  male  gametophyte,  the  process  of  fecundation  in  the  Coniferales, 
so  far  as  this  is  well  known,  is  in  general  similar  to  that  in  Cycas^ 
Zamia^  and  Ginkgo^  and  it  will  be  necessary  only  to  point  out  briefly 
the  more  important  features  of  difference. 

Since  the  important  researches  of  Strasburger,  Goroschankin,  and 
Belajeff  upon  certain  of  the  higher  Gymnosperms,  an  interesting  series 
of  facts  has  been  collected  by  Dixon  ('94),  Blackman  ('98),  Cham- 
berlain ('99),  Murrill  (1900),  Ferguson  ('01),  and  others.  The  studies 
of  later  observers,  who  used  more  improved  technique,  have  been 
confined  principally  to  the  genera  Pinus,  Picea,  and  Tsuga,  and 
consequently  our  knowledge  of  the  sexual  process  in  many  other 
Gymnosperms  is  sadly  wanting. 

It  has  been  shown  by  Strasburger  ('92)  and  others  that  the  prothal- 
lial cell  in  the  ripe  microspore  of  Pinus  and  other  closely  related  genera 
is  the  last  one  of  a  series  of  two  or  three  cells,  and  that  this  cell  divides, 
as  in  Cycas  and  Ginkgo^  to  form  the  stalk  cell  and  the  generative  cell 
of  the  antheridium  (Fig.  68,  A,  B).  The  generative  cell  (body  cell) 
then  divides  to  produce  the  two  non-motile  male  gametes,  each  consist- 
ing of  a  nucleus  surrounded  by  a  specially  differentiated  mass  of  cyto- 
plasm (Fig.  68,  C). 

Contrary  to  Cycas^  Zamia^  and  Ginkgo^  the  distal  end  of  the  male 
gametophyte,  or  pollen  tube,  grows  in  a  more  or  less  direct  line  from 
the  pollen  chamber  down  through  the  nucellus  to  the  archegonium, 
and  while  the  tube  seems  to  be  merely  a  carrier  of  the  gametes,  it  can  and 
doubtless  does  act  as  an  absorber  of  nutriment  as  well.  The  probable 
need  of  less  food  by  the  male  gametophyte  of  the  higher  gymnosperms 
may  account  for  the  absence  of  a  specially  developed  absorbing  appa- 
ratus. This  idea  is  advanced  merely  as  a  suggestion  and  not  as  an 
adequate  explanation  of  the  difference  between  the  behavior  of  the 
tube  of  Pinus^  for  example,  and  that  of  Cycas  or  Ginkgo.  Other 
factors  may  have  been  more  influential  during  the  phylogenetic  develop- 
ment of  these  forms. 


164 


ARCHEGONIATES . 


The  development  of  the  archegonium  is  the  same  as  in  the  lower 
gymnosperms.  The  ventral  canal-cell  is  separated  from  the  egg  merely 
by  a  plasma  membrane,  which  is  formed  by  the  connecting  fibers,  as 
is  usual  in  the  higher  plants.     It  persists  for  a  short  time  only.     In 


Fig.  68. — Pollen  grain,  end  of  pollen  tube,  and  fusion  nucleus  of  Pinus  itrobtu. — (After  Ferguson.) 

A,  mature  pollen  grain.    /^  and/',  remains  of  first  and  second  prothallial  cells ;  a.  c,  antheridial  cell. 

B,  pollen  grain  in  which  antheridial  cell  has  divided,    /.c,  generative  cell ;  st.c,  stalk  cell. 

C,  distal  end  of  pollen  tube  which  is  pushing  between  neck-cells  of  archegonium ;  the  male  nuclei  (x.M.) 

are  of  unequal  size,     v.n.,  tube  nucleus ;  st.c,  stalk  cell ;  s.c.,  cytoplasm  of  generative  cell. 

D,  first  mitosis  following  fecundation.     The  spindle  is  formed,  but  the  male  and   female  chromatin 

spirems  are  still  separate  and  distinct. 

Pinus  strobus^  according  to  Ferguson,  there  are  probably  instances 
in  which  the  nucleus  of  this  cell  is  not  reconstructed,  and  this  may  be 
true  also  in  other  genera  and  species. 


GYMNOSPERMS.  165 

FECUNDATION. 

Goroschanken  ('83)  observed  in  Pinus  pumilio  that  both  male  nuclei 
pass  into  the  egg-cell,  and  the  same  fact  was  established  for  Picea 
vulgaris  by  Strasburger  ('84).  Dixon  ('94)  seems  to  have  been  the 
first  to  observe  that  in  Pinus  sylvestris  all  four  nuclei  in  the  pollen 
tube,  /.  e.,  the  two  male  nuclei,  the  stalk-cell  nucleus,  and  the  tube 
nucleus  pass  into  the  egg-cell  of  the  archegonium.  This  fact  has  been 
confirmed  by  Blackman  ('98)  for  Pinus  sylvestris^  by  Murrill  (1900) 
for  Tsuga  canadensis^  and  by  Ferguson  for  Pinus  strobus.  Accord- 
ing to  Blackman  the  behavior  of  the  four  nuclei  in  Pinus  sylvestris 
can  be  easily  followed  after  their  entrance  into  the  egg-cell.  The  two 
male  nuclei  around  which  the  cytoplasm  of  the  generative  cell  can  be  no 
longer  observed  are  distinguished  by  their  larger  size.  In  P.  strobus 
one  of  these  nuclei  is  sometimes  larger  than  the  other  (Fig.  68,  C). 
The  nuclei  of  the  stalk  cell  and  tube  are,  however,  similar,  and  can 
scarcely  be  distinguished  from  each  other. 

Within  the  egg  one  of  the  two  male  nuclei  moves  toward  the  nucleus 
of  the  egg,  the  other  three  nuclei  remaining  near  the  upper  end  of  the 
cell.  On  its  way  through  the  cytoplasm  of  the  &^^  the  functional 
male  nucleus  increases  in  size,  and  in  some  cases  in  substances  stain- 
ing more  readily,  but  in  others  the  increase  in  size  seems  to  be  due  to 
vacuolation.  The  nucleus  of  the  egg-cell  in  Pinus  sylvestris  at  the 
time  of  fecundation  presents  a  strikingly  peculiar  structui-e,  which 
differs  from  that  of  the  female  nucleus  in  all  other  plants.  After  the 
formation  of  the  ventral  canal-cell  the  female  nucleus  migrates  toward 
the  center  of  the  cell,  and,  by  the  time  it  has  reached  the  middle,  it 
has  attained  an  enormous  size,  and  there  is  developed  within  it  a  rather 
coarse,  uniform,  and  wide-meshed  linin  reticulum  which  persists  until 
a  later  stage  (Fig.  69,  A) .  Within  this  linin  reticulum  the  chromatin 
is  distributed  in  irregular  masses  of  varying  size.  These  masses  may 
be  in  the  form  of  irregular  lumps  as  if  composed  of  an  aggregate  of 
granules,  or  in  shreds  or  rods  with  uneven  edges.  Sometimes  they 
appear  globular  as  small  nucleoli.  In  fact  it  is  quite  difficult  to  distin- 
guish between  some  of  the  small  nucleoli  and  similar  chromatin  masses, 
if,  indeed,  a  difference  really  exists.  The  quantity  of  chromatin  in  the 
nucleus  is  proportionally  very  small.  In  addition  to  the  linin  reticulum 
there  is  also  present  a  fine  granular  substance  which  appears  to  be 
evenly  distributed  in  the  nucleus  or  aggregated  along  the  linin  threads. 
In  the  former  case  the  nucleus  appears  more  uniformly  granular,  and  its 
linin  reticulum  stands  out  less  sharply.  The  structure  of  the  egg- 
nucleus  in  Pinus  sylvestris^  as  described  by  Blackman,  agrees  with 


1 66 


ARCHEGONIATES. 


that  of  my  own  observations,  and  from  the  work  of  Chamberlain  ('99) 
on  Pinus  laricio  and  Murrill  (1900)  on  Tsuga  canadensis^  it  seems 
that  a  similarly  constructed  nucleus  is  present  in  these  species.  In 
Pinus  strobus  (Ferguson,  '01)  the  structure  of  the  egg-nucleus  may 
vary  from  a  most  delicate  network  bearing  minute  granules  to  an  intei*- 
rupted,  imperfect  reticulum  composed  of  large,  irregular,  diffusely- 


Fig.  69. — The  fusion  of  the  sexual  nuclei  in  Pinus  sylvestris. — (After  Blackman.) 

A,  egg  cell  showing  male  nucleus  (<t)  entering  female  nucleus  (b). 

B,  later  stage  in  the  fusion  of  the  sexual  nuclei,  parental  chromatin  masses  separate. 

C,  male  and  female  nuclei  fused,  multipolar  spindle  formed. 

staining  elements.     It  has  one  large,  vacuolate  nucleolus  and  a  variable 
number  of  small  nucleoli. 

The  sexual  nuclei  of  Pinus  on  coming  together  are  in  the  resting 
condition,  and  as  in  Cycas^  Ginkgo^  and  Zamia  the  male  nucleus 
penetrates  bodily  into  the  female  nucleus.  Here  also  the  male  nucleus 
seems  to  press  with  some  force  against  the  membrane  of  the  egg-nucleus, 
thereby  forming  a   concave   depression   in   the   latter  (Fig.    69,  A). 


GYMNOSPERMS. 


167 


Although  the  male  nucleus  is  almost  enclosed  by  the  female,  actual 
fusion,  according  to  Blackman,  does  not  take  place  immediately,  since 
the  membrane  of  the  male  nucleus  is  intact  (Fig,  69,  B).  The  mem- 
brane soon  disappears,  but  the  chromatin  of  the  two  nuclei  does  not  fuse 
at  this  stage  and  no  resting  fusion  nucleus  is  formed.  With  further 
development  the  chromatin  of  each  nucleus  will  give  rise  to  a  group  of 
chromosomes,  which  become  arranged  upon  the  spindle  of  the  first 
division  after  fecundation  where  they  are  seen  to  be  split  longitudinally 
(Fig.  69,  C).  As  has  been  pointed  out  for  Ginkgo  (Fig.  67,  C,  D)  the 
spindle  seems  to  arise  entirely  within  the  limits  of  the  female  nucleus. 
In  Pinus  laricio,  according  to  Chamberlain,  after  the  male  nucleus 
is  within  the  nucleus  of  the  egg,  the  chromatin  of  the  two  pronuclei 
appear  as  two  distinct  masses  in  the  spirem  stage.  Murrill  finds  that 
in  Tsuga  canadensis  both  nuclei  are  in  the  resting  condition  when 
actual  fusion  begins,  but  he  seems  to  be  of  the  opinion  that  the  identity 
of  the  male  and  female  chromatin  can  be  traced  until  the  division  of 
the  fusion  nucleus,  as  will  be  seen  from  the  following : 

The  chromatin  of  each  nucleus  collects  in  the  form  of  a  thick  knotted  thread 
near  the  center  of  the  separating  partition,  and  the  two  masses  remain  distinct 
until  the  spirem  bands  begin  to  segment.  Just  before  the  spirems  are  formed 
the  separating  membranes  disappear  and  the  nuclear  cavities  become  united. 
The  spindle  then  arises  in  a  multipolar  fashion  between  and  among  the  two 
masses,  twelve  chromosomes  being  supplied  from  the  chromatin  of  the  sperm 
and  twelve  from  that  of  the  ^gg,  as  described  by  Blackman  for  Pinus  sylvestris. 

Ferguson  finds  in  Pinus  strobus  that  the  two  sexual  nuclei  do  not 
fuse  in  the  resting  stage.  The  male  nucleus  imbeds  itself  in  the  egg- 
nucleus  but  does  not  penetrate  its  membrane.  In  each  nucleus  is  devel- 
oped a  chromatin  spirem  and  an  achromatic  reticulum.  The  nuclear 
membranes  now  disappear,  but  the  two  chromatin  groups  remain 
distinct  until  the  nuclear-plate  stage  (Fig.  68,  D). 

The  spindle  of  the  first  division  following  fecundation  always  lies  between 
the  conjugating  nuclei  and  parallel  with  the  outer,  free  surface  of  the  sperm 
nucleus.  It  is  multipolar  in  origin  and  is  probably  derived  equally  from  the 
paternal  and  maternal  nucleus.  The  spindle  fibers  appear  to  arise  by  a  re- 
arrangement of  the  achromatic  nuclear  reticula,  and  are  evidendy  not  the 
expression  of  a  special  kinoplasmic  substance. 

In  the  stage  of  the  mature  spindle  of  the  first  division  following 
fecundation  in  Pinus  ausiriaca^  the  species  examined  by  myself,  no 
distinction  whatever  could  be  recognized  between  male  and  female 
chromatin. 


l68  ARCHKGONIATES. 

If  the  results  of  the  several  observers  referred  to  in  the  preceding 
paragraphs  be  correct,  the  behavior  of  the  fusion  nucleus  in  Pinus 
differs  not  only  from  that  of  Cycas  and  Ginkgo  as  described  by  Ikeno, 
but  also  from  the  fusion  nucleus  in  all  other  plants,  a  case  described 
in  a  species  of  Spirogyra  by  Chmielewskij  excepted. 

The  fate  of  the  other  male  nucleus,  together  with  that  of  the  stalk 
cell  and  tube,  indicates  that  these  structures  are  consumed  as  nutrient 
material.  Whether  the  cytoplasm  which  is  brought  into  the  egg  with 
the  male  nucleus  or  as  a  part  of  the  spermatozoid  has  any  morpho- 
logical or  hereditary  value  must  still  remain  an  open  question. 

From  the  standpoint  of  this  work  the  development  and  union  of  the 
sexual  elements  in  the  Gnetales  are  so  imperfectly  known  that  a  dis- 
cussion of  the  subject  will  not  be  given.  The  process  of  fecundation 
in  Gnetum  gnemon  has  been  described  in  considerable  detail  by  Lotsy 
('99),  to  whose  paper  the  reader  is  referred. 


CHAPTER  VII.— ANGIOSPERMS. 

Since  the  classical  researches  of  De  Bary  ('49)  and  Strasburger  ('78, 
'79,  '84),  especially  the  latter,  the  nature  of  the  sexual  process  in  the 
Angiosperms  has  been  a  matter  of  common  knowledge  among  botanists. 
It  is  considered  beyond  the  purpose  of  this  work  to  discuss  the  subject 
historically,  and  no  attempt  will  be  made  to  present  a  summary  of  the 
various  theories  that  have  been  advanced  from  time  to  time  during  the 
past  half  century  upon  the  homologies  of  the  female  gametophyte  or 
embryo-sac.  The  view  held  here  is  that  pollen  grains  and  embryo- 
sacs  are  respectively  micro-  and  macrospores.  The  author  is  of  the 
opinion,  as  will  be  seen  from  what  follows,  that  the  preponderance  of 
morphological  and  cytological  evidence  indicates  clearly  that  the  pollen 
mother-cell  and  the  embryo-sac  mother-cell  are  undeniably  homologous 
vv^ith  the  micro-  and  macrospore  mother-cells  of  the  archegoniates. 
The  fact  that  the  embryo-sac  mother-celb  is  not  provided  with  a  special 
or  well-differentiated  cell-wall  is  almost  without  significance  in  deter- 
mining homologies. 

THE  EMBRYO-SAC  OR  FEMALE  GAMETOPHYTE. 

Although  many  variations  occur  among  Angiosperms  in  the  develop- 
nient  of  the  embryo-sac,  yet  in  the  vast  majority  of  cases  this  process 
may  be  reduced  to  two  forms  or  types.  In  the  one  case  a  readily 
distinguishable  hypodermal  cell  of  the  nucellus,  either  with  or  without 
giving  rise  to  a  tapetum,  divides  into  an  axial  row  of  four  (sometimes 
three  ?)  cells,  or  potential  macrospores,  the  lowermost  one  developing 
usually  into  the  embryo-sac.  In  the  second  case,  which  is  typified  by 
various  species  of  Lilium^  the  hypodermal  cell  becomes  at  once  the 
macrospore.  As  illustrating  these  two  types  respectively,  the  process 
of  development  will  be  described  in  Helleborus  foetidus^  one  of  the 
Ranunculaceae,  and  Lilium  martagon. 

The  macrospore  mother-cell  of  Helleborus  foetidus  increases 
greatly  in  size,  becoming  much  longer  than  broad  in  keeping  pace 
with  the  growth  in  length  of  the  nucellus.  Its  nucleus,  which  lies 
usually  in  the  upper  end  of  the  cell,  increases  in  size  simultaneously,  as  a 
preparation  for  the  first  nuclear  division.  This  period  of  growth  of  both 
cell  and  nucleus  corresponds  to  the  period  of  growth  immediately  pre- 
ceding  the  first  nuclear  division  in  the  pollen  mother-cell  (Fig.  70,  A). 
The  nucleus  now  divides,  and,  as  a  rule,  there  follows  a  division  of 


170 


ANGIOSPERMS. 


the  cell.  The  first  nuclear  division  is  heterotypic,  corresponding  in 
detail  with  the  first  karyokinesis  in  the  microspore  mother-cell  of  the 
same  plant.  The  two  resulting  cells  soon  divide  again,  thus  giving 
rise  to  the  axial  row  of  four  cells,  the  four  potential  macrospores. 
The  second  nuclear  division  is  the  same  as  the  second  division  in  the 
pollen  mother-cell.     A  phenomenon  which  sometimes  occurs  in  Helle' 

borus  (and  it  is  probable  that 
it  may  take  place  in  other  plants 
also)  furnishes  additional  evi- 
dence in  support  of  our  hy- 
pothesis, namely,  that  the  two 
divisions  in  this  hypodermal 
cell,  or  embryo-sac  mother-cell, 
are  homologous  with  the  two 
divisions  in  the  pollen  mother- 
cell.  Cell  division  may  not 
take  place  until  after  the  second 
nuclear  division,  when  the  four 
granddaughter  nuclei  will  lie  in 
the  upper  end  of  the  cell,  and 
the  cell-plates  are  laid  down 
simultaneously  (Fig.  70,  B). 
It  has  been  observed  also  that 
the  four  nuclei,  instead  of  lying 
in  one  plane  as  in  Fig.  70,  B, 
are  sometimes  arranged  in  a 
tetrad  and  connected  with  each 
other  by  a  system  of  kinoplas- 
FiG.  70.-Embryo.sac  mother-cell  of  Heiuborus         ^^^  connecting  fibers,  as  in  the 

A.  Upper  portion  of  mother-cell  showing  nucleus  in  the      Corresponding  Stage  of  the  pol- 

prophase  of  the  first  mitosis.  Igj^  mothcr-Cell. 

B,  same  less  highly  magnified,  showing  the  four  poten-  n        <•      i  •    i 

tial  macrospores  ;  in  this  case  cell-division  did  not  J-  he    lower    Cell    Of    the    axial 

follow  first  mitosis,  and  the  plasma  membranes  mark-      row    beCOmCS     aS    a    rule     the 
ing  out  the  four  cells  were  formed  simultaneously.      ,  .  ,  _     . 

functional  macrospore.  It  m- 
creases  rapidly  in  size  at  the  expense  of  the  other  three  cells  and  the 
adjacent  tissue  of  the  nucellus,  and  develops  in  the  usual  way  into  the 
embryo-sac. 

The  unmistakable  homology  of  the  macrospore  mother-cell  of  the 
Angiosperms  with  that  of  the  Gymnosperms  has  been  very  clearly 
shown  by  Juel  (1900).  This  author  finds  in  Larix  that  the  first  and 
second  nuclear  divisions  in  the  macrospore  mother-cell,  which  give 


THE    EMBRYO-SAC    OR    FEMALE    GAMETOPHYTE. 


171 


rise  to  the  axial  row  of  four  cells,  correspond,  as  in  other  Gymno- 
sperms,  precisely  with  the  first  and  second  divisions  in  the  microspore 
mother-cell  of  this  plant.  In  my  own  opinion  the  only  legitimate 
conclusion  to  be  drawn  from  this  morphological  and  cytological  evi- 
dence is  that  the  macrospore  mother-cell  of  Larix  is  homologous  with 
that  of  Helleborus  and  other  Angiospenijs  in  which  the  embryo-sac 
develops  similarly. 

In  the  development  of  the  embryo  sac,  as  typified  by  Lilium  and 
many  other  monocotyledonous  plants,  the  hypodermal  cell  does  not 
produce  an  axial  row  of  four  cells,  but  becomes  at  once  the  functional 
macrospore.  With  the  growth  of  the  nucellus  this  hypodermal  cell 
increases  greatly  in  size,  as  does  also  its  nucleus  (Fig.  71).  The 
nucleus,  after  its  characteristic  period  of 
growth,  divides  heterotypically.  The  two 
resulting  daughter-nuclei  lie  in  the  ends  of 
the  cell.  No  cell-division  follows  this 
nuclear  division,  although  the  thickening  of 
the  connecting  fibers  in  the  equatorial  region 
seems  to  indicate  that  a  tendency  toward  cell- 
division  existed  (Fig.  72,  A).  The  macro- 
spore continues  its  growth,  and  the  daughter- 
nuclei  divide.  This  division  is  homotypic 
and  corresponds  exactly  to  the  second  mitosis 
in  the  pollen  mother-cell.  The  four  resulting 
nuclei  have,  as  a  rule,  the  orientation  shown 
in  Fig.  72,  B.  Very  frequently  no  vacuole 
is  present  at  this  stage,  and  the  four  nuclei  are 
connected  with  each  other  and  with  the  plasma 
membrane  by  systems  of  kinoplasmic  radiations  and  connecting  fibers. 
The  increase  of  the  cell  in  length  is  now  rapid,  and,  as  a  result,  one 
or  more  large  vacuoles  are  formed  at  the  center  or  near  the  micropylar 
end  of  the  sac.  Two  of  the  four  nuclei  which  are  sisters  move  into 
the  upper,  and  the  other  two  into  the  lower  end  of  the  cell.  In  normal 
cases  the  nuclei  in  each  end  divide  so  that  a  group  of  four  nuclei  occu- 
pies each  end.  The  four  nuclei  in  the  micropylar  end  are  arranged 
either  in  a  plane,  or  nearly  so,  or  in  the  form  of  a  tetrad  (Fig.  73, 
A,  B).  The  arrangement  and  behavior  of  the  nuclei  in  the  chalazal 
end  of  the  sac  is  more  variable  (Mottier,  '97). 

As  a  rule  the  two  nuclei  in  the  micropylar  end  of  the  sac,  and  it  is 
with  these  that  we  are  especially  concerned,  divide  simultaneously,  and, 
before  cell-plates  are  laid  down,  the  four  resulting  nuclei  are  connected 


Fig.  71  — Embryo-sac  mother-cell 
oi  Lilium  martagon  with  nu- 
cleus showing  beginning  of 
prophase  of  division. 


172 


ANGIOSPERMS. 


by  beautiful  systems  of  kinoplasmic  connecting  fibers.  Cell-plates,  or 
plasma  membranes,  are  next  formed  by  the  connecting  fibers,  in  a  man- 
ner common  to  the  higher  plants,  by  which  the  three  cells  of  the  egg- 
apparatus  are  differentiated,  while  a  fourth  nucleus,  the  upper  polar 
nucleus  and  a  sister  of  the  egg-nucleus,  remains  free  in  the  cytoplasm 
(Fig.  73,  B).  In  A,  Fig.  ,73?  three  nuclei  of  the  tetrad  are  shown. 
The  cell-plates  are  nearly  formed,  and  it  is  clear  that  the  lower  cell  to 
the  right  will  become  the  egg-cell,  while  the  nucleus  to  the  left  is 


Fig.  •J2, — Later  stages  in  development  of  embryo-sac  of  Liliunt  ntarta^on. 

A,  the  nucleus  hat  divided ;  the  daughter-nuclei  are  connected  by  connecting  fibers 
which  have  shown  a  tendency  to  form  a  cell-plate. 

B,  close  of  second  nuclear  division ;  the  four  nuclei  are  connected  with  each  other 

and  with  plasma  membrane  by  kinoplasmic  fibers, 

unquestionably  the  upper  polar  nucleus.  The  cytoplasm  immediately 
surrounding  this  nucleus  is  not  delimited  by  a  plasma  membrane  as  in 
the  case  of  the  other  three  cells.  In  B,  Fig.  73,  the  relation  of  all 
four  nuclei  is  evident. 

The  antipodal  cells  in  Lilium  martagon  are  formed  in  the  same 
way  as  those  of  the  egg-apparatus  when  the  process  is  normal,  although 
the  development  of  these  cells  is  not  infrequently  variable  in  this 
species  (Mottier,  '97).  Among  the  Angiosperms  in  general  the  anti- 
podal cells  represent  a  very  variable  group  both  as  to  number  and 


THE  EMBRVO-SAC  OR  FEMALE  GAMETOPHYTE.        1 73 

period  of  duration.  In  many  plants  they  disorganize  immediately 
after  they  are  formed ;  in  others  they  may  divide  repeatedly,  giving 
rise  to  a  larger  or  smaller  mass  of  tissue  which  remains  functional  for 
a  comparatively  long  time.  The  development  of  the  antipodal  cells 
into  a  mass  of  tissue,  whose  fuHction  is  probably  concerned  with  the 
absorption  and  elaboration  of  food  materials,  may  occur  in  the  most 
widely  separated  families — a  fact  which  goes  to  show  that  this  phe- 
nomenon is  a  special  adaptation  in  each  specific  case  and  in  no  way 
indicative  of  a  closer  phylogenetic  lelationship  or  a  primitive  condition. 

The  typical  embryo-sac,  or  female  gametophyte,  consists,  therefore, 
of  seven  cells,  one  of  which,  the  egg-cell,  is  the  female  gamete,  while 
the  other  cells  may  be  looked  upon  as  vegetative  or  prothallial  cells 
(Fig.  73,  C).  The  egg-cell  may  be  regarded  as  the  homologue  of  the 
egg-cell  in  the  Gymnosperms,  and  hence  a  rudimentary  archegonium. 
Whether  the  synergidse  are  to  be  regarded  as  rudimentary  egg-cells,  or 
merely  prothallial-cells,  can  not  be  determined  at  the  present  state  of 
our  knowledge. 

As  stated  in  a  preceding  paragraph,  no  attempt  will  be  made  even 
to  summarize  the  numerous  variations  in  the  development  of  the 
embryo-sac  that  have  been  observed  by  the  many  investigators,  since 
the  vast  majority  of  these  variations  may  reasonably  be  considered  as 
special  adaptations,  and  as  such  are  of  small  theoretical  importance. 

One  of  the  many  interesting  cases  about  which  there  is  likely  to  be 
much  diversity  of  opinion  will  be  briefly  mentioned.  This  is  found 
in  the  development  of  the  embryo-sac  of  Peperomia  pellucida^  as 
described  by  Campbell  ('99,  '01)  and  Johnson  (1900).  In  this  species 
sixteen  nuclei  are  present  in  the  mature  embryo-sac.  Of  these  one 
becomes  the  nucleus  of  the  ^^g^  one  the  single  synergid,  and  several, 
usually  eight,  fuse  to  form  the  endosperm  nucleus.  The  remaining 
nuclei,  according  to  Johnson,  degenerate,  but  Campbell  finds  that  they 
are  scattered  in  the  sac,  each  developing  about  itself  a  cell-wall  much 
as  do  the  antipodal  cells  of  many  Angiosperms.  Johnson  regards  the 
peculiarities  of  the  embryo-sac  in  Peperotnia  as  secondarily  acquired 
from  the  typical  form,  while  Campbell  looks  upon  them  as  primitive, 
recalling  such  forms  among  the  Gymnosperms  as  Gnetum  gnemon 
(Lotsy,  1900). 

In  the  development  of  the  embryo-sac,  as  typified  by  Lilium^  the 
two  cell-divisions  which  result  in  the  axial  row  of  four  cells  in  Helle- 
borus  are  wanting,  and  the  question  arises  whether  the  hypodermal 
cell  of  Lilium^  for  example,  which  develops  directly  into  the  embryo- 
sac,  is  homologous  with  the  hypodermal  cell  of  Helleborus^  or  only 


174 


ANGIOSPERMS. 


with  that  one  of  the  axial  row  which  develops  into  the  embryo-sac. 
The  view  held  by  the  author  is  that  the  hypodermal  cells  in  both  cases 
are  macrospore  mother-cells.  In  Lilium  this  maci-ospore  mother-cell 
becomes  at  once  the  macrospore,  while  in  Helleborus  it  gives  rise  to 
four  spores.  In  both  cases  the  reduoed  number  of  chromosomes  is 
present,  and  the  egg-cell  of  Lilium  is  hereditarily  the  equivalent  of 
the  egg-cell  in  Helleborus.  The  number  of  cell-divisions  elapsing 
between  that  period  in  which  the  reduced  number  of  chromosomes 
appears  and  the  differentiation  of  the  sexual  cells  is  of  no  importance, 
since  in  many  ferns,  for  example,  thousands  of  cell-divisions  occur 
between  these  points  in  ontogeny.  It  seems,  therefore,  that  the  view 
held  here  not  only  does  no  violence  to  either  the  facts  of  morphology 
or  cytology,  or  to  the  most  widely  accepted  theory  concerning  the 
significance  of  the  reduction  of  the  number  of  chromosomes,  but  it  is 
also  in  complete  harmony  with  these  facts. 

THE  MALE  GAMETOPHYTE. 

As  in  the  case  of  the  embryo-sac,  the  development  of  the  male 
gametes  in  the  microspore  or  in  the  pollen  tube,  the  male  gameto- 
phyte,  is  so  well  known  that  only  the  briefest  mention  of  it  is  necessary. 

In  the  microspore  of  Lilium^  in  which  the  cytological  details  are 
probably  best  understood,  the  antheridial  or  generative  cell  is  clearly 
differentiated  from  the  remaining  cytoplasm  of  the  spore  by  a  plasma 
membrane.  The  generative  cell  is  moon-shaped  or  crescenticin  Z?7/mz« 
candidum  and  L.  martag-on,  and  its  cytoplasm  behaves  somewhat 
differently  toward  certain  stains,^  so  that  the  contrast  between  the  gen 
erative  cell  and  the  cytoplasm  of  the  tube  cell  is  often  very  striking. 
Strasburger  ('98),  who  attributes  a  fibrillar  structure  to  the  cytoplasm 
of  the  generative  cell,  regards  it  as  kinoplasm,  and  since  some  cyto- 
plasm accompanies  the  male  nucleus  into  the  embryo-sac,  the  theory 
may  not  be  without  significance.  In  Lilium  and  in  many  other 
Angiosperms  the  generative  or  antheridial  cell  divides  in  the  pollen 
tube  to  give  rise  to  the  two  male  gametes,  but  in  some  instances  this 
division  takes  place  in  the  spore.  Each  male  gamete  consists,  there- 
fore, of  a  nucleus  surrounded  by  a  small  portion  of  cytoplasm  derived 
from  the  generative  cell. 

Nothing  need  be  added  here  concerning  the  growth  of  the  pollen 
tube  toward  the  egg-cell  of  the  embryo-sac.  The  result  is  the  same 
whether  the  tube  enters  through  the  micropyle  or  chalaza.  The  end 
of  the  tube  may  enter  the  sac  at  one  side  of  one  of  the  synergidae,  in 


'  E.  /.,  Flemming's  triple  stain. 


THE    MALE    GAMETOPHYTE. 


175 


which  case  only  one  of  these  cells  is  at  once  disorganized,  the  other 
retaining  its  normal  structure  for  some  time,  or  it  may  enter  between 
the  two  synergidse,  when  both  cells  are  destroyed  almost  immediately. 


^  fyrt 


-  pn^ 


oa-^t.b. 


>  cuvt 


Fig.  73.— Formation  of  egg-apparatus  and  mature  embryo-sac  in  Lilium  martagan. 

A,  telophase  of  third  mitosis;  the  four  nuclei,  three  only  shown,  form  a  tetrad;  the  lower  nucleus  to 

the  right  is  the  egg-nucleus,  the  one  to  left  the  upper  polar  nucleus  ;  plasma  membranes  delimiting 
the  three  cells  of  egg-apparatus  are  just  formed. 

B,  same  stage,  perhaps  a  little  later,  showing  all  four  nuclei  in  a  plane  ;  the  lower  nucleus  on  left  is  the 

upper  polar  nucleus. 

C,  mature  embryo-sac  into  which  the  male  nuclei  have  been  discharged.    «.».,  egg-nucleus  ;  tn.n.,  male 

nucleus  applied  to  that  of  the  egg;  m.n.',  second  male  nucleus  approaching  upper  polar  nucleus; 
syM.,  disorganized  synergid;  />.«.,  polar  nuclei;  t.i.,  trophoplasmic  body;  ant.,  antipodal  cells. 

As  soon  as  the  end  of  the  pollen  tube  enters  the  embryo-sac  it 
opens,  discharging  the  two  male  gametes  and  other  contents.  One 
of  the  male  nuclei  enters  the  egg-cell  and  applies  itself  to  the  nucleus 
of  the  egg,  while  the  other  passes  on  into  the  cavity  of  the  sac  (Fig. 
73,  C).     As  soon  as  the  male  nuclei  have  been  discharged  into  the 


176 


ANGIOSPERMS. 


embryo-sac  and  can  be  distinctly  recognized,  no  trace  of  the  cytoplasm 
which  accompanied  them  in  the  tube  can  be  distinguished,  so  that 
the  exact  behavior  of  this  cytoplasm  is  unknown.  Consequently  we 
are  concerned  hei*e  solely  with  the  union  of  the  nuclei. 

THE  FUSION  OF  MALE  AND  EGG-NUCLEI, 

We  shall  follow  first  the  male  nucleus  which  fuses  with  that  of  the 
egg-cell.  It  is  presumably  the  first  male  nucleus  which  escapes  from  the 
pollen  tube  that  unites  with  the  nucleus  of  the  egg,  but  positive  proof 

on  this  point  is  want- 
ing. In  certain  spe- 
cies of  Lilium^  and 
various  observers 
have  shown  this  to  be 
true  of  many  other 
Angiosperms,  the 
male  nucleus,  when 
observed  in  the  egg- 
cell,  is  frequently 
sausage- shaped, 
worm-like,  or  S- 
shaped  (Mottier, 
'97),  making  one  or 
more  spiral  -  like 
turns,  which  is  sug- 
gestive of  a  worm- 
like motion,  but  posi- 
tive proof  of  any  such 
movement  is  want- 
ing. It  applies  itself 
to  the  nucleus  of  the  ^%g.,  retaining  the  form  mentioned  for  some  time 
(Fig.  74,  A).  The  structure  of  the  two  sexual  nuclei  at  this  stage  is 
accurately  shown  for  Lilium  martagon  in  this  figure.  The  two 
nuclei  are  in  the  resting  condition,  although  the  chromatin  of  the 
male  nucleus  is  a  little  more  regularly  arranged.  The  male  nuclei 
when  in  the  embryo-sac  stain  a  deeper  red,  safranin,  gentian  violet 
and  orange  G  being  used,  than  the  other  nuclei  of  the  sac,  and  for 
that  reason  they  may  be  readily  recognized.  As  fusion  progresses,  the 
nuclei  become  quite  alike  in  shape,  size  and  structure  (Fig.  74,  B). 
Their  membranes  gradually  disappear  at  the  place  of  contact,  their 
cavities  become  one,  and  the  resulting  fusion  nucleus,  which  is  in  the 


Fig.  74. — Fusion  of  sexual  nuclei. 

A,  vermiform  male  nucleus  applied  to  egg-nucleus,  Lilium  martagon. 

B»  egg-cell  oi  Lilium  candidum,  showing  sexual  nuclei  in  act  effusing ; 

the  nuclear  membranes  have  disappeared  at  place  of  contact. 


FATE    OF    SECOND    MALE   NUCLEUS    IN    EMBRYO-SAC.  1 77 

resting  condition,  can  scarcely  be  distinguished  from  the  nucleus  of 
an  unfecundated  egg.     The  nucleoli  finally  unite  also. 

Thfe  worm-like  or  S-shape  form  of  the  male  nucleus  in  L ilium ^ 
first  described  by  the  author  in  1897  (Mottier,  '97,  p.  23),  has  since 
that  time  attracted  the  close  attention  of  students  of  fecundation 
generally.  Guignard,  having  observed  the  same  phenomenon  in  1899, 
concluded  to  designate  these  vermiform  nuclei  as  antherozoids,  evidently 
attributing  to  them  the  power  of  locomotion.  As  a  matter  of  fact  these 
nuclei  do  not  possess  cilia  or  any  other  cytoplasmic  organ  of  loco- 
motion, nor  have  the  male  nuclei  in  any  Angiosperm  been  found  to 
possess  any  such  structures.  Nuclei  in  many  vegetative  cells  of  both 
plants  and  animals  are  known  to  be  able  to  change  their  form,  and  the 
fact  that  in  the  embryo-sac  the  male  nuclei  may  assume  a  worm-like 
shape,  which  merely  suggests  a  squirming  or  vermiform  motion,  is  not 
a  sufficient  reason  for  designating  them  as  spermatozoids.  So  far  as 
is  known,  all  spermatozoids  are  provided  with  a  cytoplasmic  organ  of 
locomotion,  existing  in  the  form  of  a  cilium  or  cilia,  and  it  certainly 
does  not  conduce  to  clearness  to  apply  this  term  to  the  male  nuclei  of  the 
Angiosperms.  Strasburger  (1900)  claims  that  the  vermiform  nucleus 
moves  passively  in  the  embryo-sac,  basing  his  opinion  upon  observa- 
tions of  the  embryo-sac  of  Monotropa  in  the  living  condition.  A 
streaming  movement  was  seen  in  the  cytoplasmic  strand  connecting 
the  egg-cell  with  the  endosperm  nucleus,  and,  in  the  light  of  this  fact, 
it  is  highly  probable  that  the  second  male  nucleus  is  carried  to  the 
endosperm  nucleus  by  that  means. 

THE  FATE  OF  THE  SECOND  MALE  NUCLEUS  IN  THE 
EMBRYO-SAC. 

The  fact  that  one  of  the  male  nuclei  fuses  with  a  polar  nucleus,  or 
with  the  endosperm  nucleus  in  certain  lilies  and  in  species  of  widely 
separated  families,  has  also  aroused  a  keen  interest  among  botanists, 
and  has  called  forth  much  interesting  and  suggestive  speculation.  In 
1897  the  author  called  attention  to  the  fact  that  the  second  male  nucleus 
in  Lilium  martagon  applied  itself  to  one  of  the  polar  nuclei,  but  the 
actual  fusion  was  not  observed.  The  plants  from  which  the  material 
was  obtained  produced  few  or  no  seeds  that  year,  and  all  preparations 
of  embryo-sacs,  examined  at  a  time  when  normally  fecundated  eggs 
should  have  been  present,  gave  only  evidence  of  disorganization,  and 
it  was  concluded  that  probably  a  fusion  of  the  nuclei  did  not  proceed 
further,  which  under  the  circumstances  may  have  been  true.  Later, 
other  investigators  as  well  as  the  author  have  observed  this  nuclear 


178 


ANGIOSPERMS. 


fusion  in  species  of  Lilium  (Fig.  75,  A,  B,  C).  An  account  of  the 
fusion  of  one  of  the  male  nuclei  with  the  polar  nuclei  was  first  pub- 
lished by  Nawaschin  ('99)  and  made  known  to  botanists  in  general 
by  a  reference  in  the  Botanisches  Centralblatt. 

Guignard  ('99)  in  the  same  year  published  the  results  of  his  obser- 
vations confirming  the  statement  of  Nawaschin.  He  figured  the  second 
vermiform  male  nucleus  in  contact  with  one  or  both  polar  nuclei,  but 
none  of  Guignard's  figures  showed  an  actual  fusion.  Although  we 
are  justified  in  assuming  that  sexual  nuclei,  when  brought  in  contact, 
will  fuse,  yet  the  possibility  is  not  excluded  that  since  the  sexual  nuclei 
remain  side  by  side  for  some  time  before  fusion  takes  place,  the  causes 
w^hich  have  been  long  known  to  operate  in  preventing  the  formation 


Fig.  75. — Fusion  of  second  male  nucleus  with  polar  nuclei  in  Lilium  )Harta£on. 

A,  an  S-shaped  male  nucleus  applied  to  the  upper  polar  nucleus. 

B,  second  male  nucleus  (shown  only  in  part)  and  the  two  polar  nuclei  close  together. 

C,  all  three  nuclei  fusing. 

of  seeds  in  certain  species  of  Lilium  may  also  prevent  the  complete 
fusion  of  these  nuclei  after  having  come  in  contact. 

The  fusion  of  a  male  nucleus  with  the  endosperm  nucleus  has  received 
different  interpretations  at  the  hands  of  the  several  investigators.  Na- 
waschin (1900),  H.  De  Vries  ('99,  1900)  and  Correns  ('99)  evidently 
see  in  this  fusion  a  true  sexual  process,  basing  their  conclusion  largely 
upon  the  hybrid  character  of  the  endosperm  of  certain  varieties  of  Zea 
mays.  Guignard  in  his  paper  upon  Tulipa  celliana  and  T.  sylveS' 
iris  regards  the  process  as  a  pseudo-fecundation. 

From  a  series  of  important  experiments  on  the  hybridization  of 
several  varieties  of  Zea  mays^  Webber  (1900)  arrives  independently  at 
the  same  conclusion  as  De  Vries,  namely,  that  certain  phenomena  of 
xenia  are  the  result  of  the  fusion  of  one  of  the  male  nuclei  with  the 
endosperm  nucleus.  As  a  result  of  the  crossing,  the  endosperm,  pro- 
duced in  the  same  embryo-sac  with  the  hybrid  embryo  sporophyte, 


FATE    OF    SECOND    MALE    NUCLEUS    IN    EMBRYO-SAC.  1 79 

shows  certain  well-marked  characters  of  the  male  parent,  and  accord- 
ing to  the  hypothesis  of  Webber,  De  Vries,  and  others,  these  hybrid 
characters  are  transmitted  by  the  male  nucleus.  In  some  cases  the 
endosperm  does  not  reveal  hybrid  characters,  but  only  those  of  the 
mother  plant,  and  Webber  explains  the  fact  by  assuming  that  in  those 
cases  the  endosperm  nucleus  may  not  have  been  fecundated.  As  an 
explanation  of  another  peculiar  feature  of  xenia  in  certain  varieties  of 
maize,  which  is  shown  by  a  variegated  or  mosaic  endosperm,  Webber 
suggests  that  probably  the  second  male  nucleus  may  not  have  united 
with  the  endosperm  nucleus,  but  it  may  have  been  able  to  divide  in- 
dependently. If  this  should  occur,  there  would  then  be  formed  in  the 
embryo-sac  nuclei  of  two  distinct  characters,  one  group  from  the 
division  of  the  endosperm  nucleus  and  one  from  the  sperm  nucleus. 
Or  a  second  hypothesis  lies  in  the  probability  that  the  second  male 
nucleus  fuses  with  one  of  the  polar  nuclei,  and  that  after  fusion  the 
other  polar  nucleus  is  repelled  and  develops  independently.  In  view 
of  the  fact  that  in  the  sea-urchin  (Boveri,  '95)  the  male  nucleus  is 
capable  of  independent  division  under  certain  circumstances,  these 
hypotheses  are  certainly  very  suggestive,  but  they  have,  as  yet,  among 
plants  no  support  based  upon  observation,  especially  since  partheno- 
genesis is  unknown  in  maize.  Before  these  suggestions  can  be  of 
much  value  in  explaining  the  phenomenon,  it  is  necessary  to  know 
whether  a  male  nucleus  is  of  itself  capable  of  division  in  the  embryo- 
sac,  and  whether  one  of  the  polar  nuclei  without  having  united  with 
the  other  or  with  a  sperm  nucleus  is  also  capable  of  independent 
division. 

Although  the  union  of  a  male  nucleus  with  the  endosperm  nucleus 
may  be  conclusively  shown  to  be  the  cause  of  hybrid  endosperm  in  maize, 
yet  that  fact  alone  is  not  sufficient  to  justify  the  unqualified  conclusion 
that  the  fusion  represents  a  real  fecundation.  Strasburger,  in  discus- 
sing this  question  at  some  length  in  the  Botanische  Zeitung  (pp.  293- 
316,  1900),  argues  forcibly  against  the  doctrine  of  a  double  sexual 
process  as  understood  by  Nawaschin,  and  proposes  a  different  interpre- 
tation of  the  two  sets  of  nuclear  fusions.  For  the  union  of  the  male 
nucleus  and  that  of  the  egg-cell  which  results  in  an  individual  sporophyte, 
the  expression  generative  fecundation  is  used,  while  the  fusion  of  the 
other  male  nucleus  with  the  endosperm  nucleus  is  designated  vegetative 
fecundation.  In  the  interpretation  of  Strasburger,  the  need  of  genera- 
tive fecundation  by  means  of  sexual  nuclei  of  different  origin  lies  in  the 
equalization  of  individual  variations,  which  is  necessary  for  the  continu- 
ance of  the  species,  while  in  vegetative  fecundation  there  is  merely  the 


l8o  ANGIOSPERMS. 

manifestation  of  a  growth  stimulus.  Vegetative  fecundation  according 
to  this  interpretation  finds  its  parallel  in  such  phenomena  as  described  by 
Klebs  ('98,  1900),  Loeb  ('99,  '01)  and  Nathansohn  (1900),  in  which, 
by  means  of  physical  or  chemical  stimuli,  such  as  increased  tempera- 
ture or  an  increase  of  the  osmotic  power  of  the  surrounding  fluid, 
unfecundated  egg-cells  have  been  made  to  develop  parthenogenetically 
through  certain  embryonic  stages.  According  to  the  view  of  Stras- 
burger,  therefore,  sexual  reproduction  embraces  fundamentally  two 
great  and  far-reaching  factors,  namely,  the  union  of  hereditary  ele- 
ments and  the  imparting  of  a  growth  stimulus.  In  the  fusion  of  a 
male  nucleus  with  the  endosperm  nucleus,  only  one  of  these  factors, 
the  stimulus  to  growth,  is  manifested,  since  the  interrupted  growth  of 
the  endosperm  is  enabled  to  continue.  The  result  is  the  same  whether 
the  second  sperm  nucleus  unites  with  the  endosperm  nucleus  or  not, 
and  furthermore  because  the  endosperm  is  not  an  individual  in  the 
sense  that  the  embryo  sporophyte  is  an  individual.  It  is  further  true 
that  the  endosperm  nucleus  may  divide  and  give  rise  to  several  nuclei 
before  the  contents  of  the  pollen  tube  are  discharged  into  the  embryo- 
sac,  and  in  case  that  no  pollen  tube  reaches  the  embryo-sac,  these  same 
endosperm  nuclei  never  continue  their  development.  It  is  reasonable 
to  conclude,  therefore,  that  a  growth  stimulus  may  be  imparted  to 
the  endosperm  by  the  act  of  fecundation  in  the  egg-cell,  just  as  the 
vegetative  tissue  of  certain  parts  of  the  pistil  are  stimulated  to  growth 
by  the  presence  of  the  pollen  tube. 

Many  who  agree  with  Strasburger  may  probably  not  consider  it 
necessary  or  advisable  to  use  the  term  "  vegetative  fecundation."  The 
author  does  not  see  the  necessity  of  associating  the  idea  of  fecundation 
with  this  process  of  nuclear  fusion,  for  the  reason  that  nuclear  fusions 
in  vegetative  cells  do  not  signify  an  act  of  fecundation.  In  the  light 
of  all  the  known  facts,  it  seems  that  we  have  to  do  here  with  purely 
vegetative  fusions,  and  that  we  are  not  justified  in  attributing  to  such 
nuclear  fusions  the  idea  of  sexuality.  Although  the  upper  polar 
nucleus  is  the  sister  of  the  egg-nucleus,  it  does  not  necessarily  follow 
that  the  former  is  also  a  female  nucleus,  since  it  is  certainly  not  true  that 
the  sister  cells  of  egg-cells  are  even  potential  gametes.  If  such  an 
assumption  were  accepted,  then  the  ventral  canal-cell  of  the  arche- 
goniates  might  be  considered  an  egg-cell,  a  doctrine  to  which  the 
author  can  not,  as  yet,  subscribe. 


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